Hypoxia inducing factors and uses thereof for inducing angiogenesis and improving muscular functions

ABSTRACT

This invention provides HIF-3α nucleic acid and sequences. Also provided are methods for using HIF-3α nucleic acids, proteins, fragments, antibodies, probes, and cells, to characterize HIF-3α, modulate HIF-3α cellular levels, induce angiogenesis, improve muscular function, and treat coronary and cardiac diseases in mammals.

RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Applications60/292,630 filed May 23, 2001, and 60/354,529 filed Feb. 8, 2002, thedisclosures of which are incorporated by reference herein in theirentireties.

BACKGROUND OF THE INVENTION

[0002] a) Field of the Invention

[0003] The present invention is concerned with a human protein called“HIF-3α” that is a Hypoxia Inducible Factor-3α and more particularly tothe use of HIF-3α nucleic acids, proteins, fragments, antibodies,probes, and cells, to characterize HIF-3α, and modulate its cellularlevels.

[0004] The present invention is also concerned with the use ofnucleotide sequences encoding for proteins from the hypoxia induciblefactors family for inducing VEGF expression, for inducing angiogenesisand for improving muscular functions, and more particularly, fortreating coronary and cardiac diseases in mammals.

[0005] b) Brief Description of the Prior Art

[0006] Chronic ischemic heart disease is a worldwide health problem ofmajor proportions. According to the American Heart Association, 61 800000 Americans have at least one type of cardiovascular disease. Inparticular, coronary heart disease (CHD) cause myocardial infarction(MI) for 7 500 000 American patients and congestive heart failure (CHF)for 4 800 000 American patients. Almost 450 000 deaths in the UnitedStates alone were deemed to derive from CHD.

[0007] Current CHD treatments include medication, percutaneoustransluminal coronary angioplasty and coronary artery bypass surgery.These procedures are quite successful to increase blood flow in themyocardium thus reducing ischemia and ameliorating the condition of thepatient. However, due to the progressive nature of CHD, the beneficialeffects of these procedures are not permanent and new obstructions canoccur. Patients that live longer through effective cardiovascularinterventions eventually run out of treatment options. Also an importantpatient population is still refractory to these treatments due todiffuse athereosclerotic diseases and/or small caliber arteries.

[0008] Severe and chronic ischemia can cause MI which is an irreversiblescarring of the myocardium. This scarring reduces heart contractilityand elasticity and consequently the pumping function, which can thenlead to CHF. Treatments available to CHF patients target kidney functionand peripheral vasculature to reduce the symptoms but none are treatingthe scar or increasing pump function of the heart. A very promisingapproach for reducing the scar and improving heart function is namedcellular cardiomyoplasty (CCM). It consists in the injection of cells inthe scar, replacing the fibrotic scar by healthy tissue and increasingelasticity (see U.S. Pat. Nos. 5,130,141; 5,602,301, 6,099,832 and6,110,459).

[0009] Another emerging treatment for CHF patients is therapeuticangiogenesis. Angiogenesis is defined as blood vessel sprouting andproliferation from pre-existing vasculature. The net result is a highercapillary density and better blood perfusion. For instance, U.S. Pat.No. 5,792,453 disclose a method for promoting coronary collateral vesseldevelopment by delivering an adenovirus vector with a transgene encodingfor an angiogenic protein. Although stimulation of angiogenesis canimprove function of ischemic myocardium, it will have no effect on scartissue because no viable cells will benefit from the improved perfusion.

[0010] Many growth factors are currently used to induce angiogenesis,including Vascular Endothelial Growth Factor (VEGF) and FibroblastGrowth Factor (FGF), but none of these factors has the property tostimulate every step of angiogenesis (basal membrane disruption,endothelial cell proliferation, migration and differentiation followedby periendothelial cells recruitment). Since it is known that cellhypoxia can naturally induce a strong angiogenesis, the use ofregulators of hypoxia could stimulate the synthesis of one or manyangiogenic factors at once, thereby resulting in a more structured andstronger angiogenesis than with individual factors.

[0011] Hypoxia Inducible Factors (HIFs) are heterodimeric transcriptionfactors that regulate a number of adaptive responses to low oxygentension. They are composed of alpha- and beta-subunits that belong tothe basic helix-loop-helix-PAS (bHLH-PAS) superfamily. Members of thisfamily include HIF-1α (also known as MOP1; see Wang et al., Proc. Natl.Aca. Sci. USA (1995) 92:5510-5514; and U.S. Pat. Nos. 5,882,314;6,020,462 and 6,124,131), HIF-2α (also known as Endothelial PAS 1(EPAS1), MOP2, HIF-related factor (HRF) and HLF (HIF-like factor), seeTian et al., Genes & Dev. (1996) 11:72-82; and U.S. Pat. No. 5,695,963).

[0012] Another member of the HIF family has been discovered recently,namely HIF-3α. The cloning of HIF-3α has been described in mice (Gu etal., Gene Expression (1998) 7:205-213; and in International PCTapplication WO 99/28264) and in rat (Kietzmann et al., Biochem J.(2001), 354 (Pt3):531-537). A partial cDNA sequence of human HIF-3α hasbeen published in 1999 (GenBank™ accession No. AF079154), and a fulllength sequence of a human HIF-3α isoform, different from the one of thepresent invention, was published in October 2001 by Hara et al.(Biochem. Biophys. Res. Comm. (2001), Oct 5; 287:808-813).

[0013] HIFs are highly labile in normal conditions, but are stabilizedin response to low oxygen tension. This stabilization allows them tobind to cis DNA elements of target genes, and stimulate transcription ofhypoxia induced genes that help cell survival in low oxygen conditions.These target genes are implicated in processes such as anaerobicmetabolism (glucose transporters and glycolytic enzymes), vasodilatation(inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1)),increased breathing (tyrosine hydroxylase), erythropoiesis(erythropoietin) and angiogenesis (VEGF). Gene activation by HIF-1α orHIF-2α was demonstrated by co-transfection assays, in which a reportergene is activated by the co-transfected HIF factor (Tian et al., Genes &Dev. (1996) 11: 72-82; Jiang et al., J. Biol. Chem. (1995) 272:19253-19260). The role of HIF-2α in VEGF activation was alsodemonstrated in renal cell carcinoma (Xia et al., Cancer (2001),91:1429-1436). In animal models, strong angiogenesis was reportedfollowing gene transfer of a hybrid HIF-1α/VP16 DNA construct (Vincentet al., Circulation (2000) 102: 2255-2261). However, prior to thepresent invention, it has never been demonstrated or suggested thatHIF-2α or HIF-3α could induce the expression of angiogenesis-relatedgene(s) in mammalian muscular cells, nor that they could induceangiogenesis in these cells. It was also unknown that expression ofHIF-1α, HIF-2α or HIF-3α in ischemic muscular tissue resulted in anincreased metabolic activity of this tissue, indicating improvedfunction.

[0014] Given that HIFs seem to represent ideal factors for VEGFactivation and/or for induce angiogenesis, there is thus a need toidentify a novel member of the HIF family. There is more particularly aneed for a human HIF-3α protein and a nucleic acid encoding the same.

[0015] Also, it would be highly desirable to be provided with methods,compositions and cells for inducing angiogenesis and for improvingmuscular functions.

[0016] The present invention fulfils these needs and also other needs asit will be apparent to those skilled in the art upon reading thefollowing specification.

SUMMARY OF THE INVENTION

[0017] The present inventors have discovered a novel member of the humanHypoxia Inducible Factors (HIFs) HIF-3α. The present inventors have alsodiscovered uses for human HIF-3α proteins, fragments, nucleic acids, andantibodies for modulating HIF-3α cellular levels, for inducing VEGFexpression in a mammalian cell, and for inducing angiogenesis in amammalian tissue.

[0018] In general, the invention features an isolated or purifiednucleic acid molecule, such as genomic, cDNA, antisense, DNA, RNA or asynthetic nucleic acid molecule that encodes or corresponds to a humanHIF-3α polypeptide.

[0019] According to a first aspect, the invention features isolated orpurified nucleic acid molecules, polynucleotides, polypeptides, humanHIF-3α proteins and fragment thereof. Preferred nucleic acid moleculesconsist of a cDNA.

[0020] In a first embodiment, the isolated or purified nucleic acidmolecule encodes a human protein that has the biological activity of ahuman HIF-3α polypeptide.

[0021] According to a specific embodiment, the nucleic acid of theinvention comprises a sequence selected from the group consisting of:

[0022] a) sequences provided in SEQ ID NO: 1 or 3;

[0023] b) complements of the sequences provided in SEQ ID NO: 1 or 3;

[0024] c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NO: 1 or 3;

[0025] d) sequences that hybridize to a sequence provided in SEQ ID NO:1 or 3, under moderately or strong stringent conditions;

[0026] e) sequences having at least 75% identity to a sequence of SEQ IDNO: 1 or 3; and

[0027] f) degenerate variants of a sequence provided in SEQ ID NO: 1 or3.

[0028] More preferably, the nucleic acid molecule of the inventioncomprises a sequence selected from the group consisting of:

[0029] a) a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%or 97% nucleotide sequence identity with SEQ ID NO: 1; and

[0030] b) a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%or 97% nucleotide sequence identity with a nucleic acid encoding anamino acid sequence of SEQ ID NO: 2.

[0031] Even more preferably, the nucleic acid molecule comprises asequence substantially the same or having 100% identity with SEQ ID NO:1 or a sequence substantially the same or having 100% identity withnucleic acids encoding an amino acid sequence of SEQ ID NO: 2. Mostpreferred nucleic acid molecules are those comprising part or all ofnucleic acids 1423 to 1545 of SEQ ID NO: 1, and/or those comprising partor all of nucleic acids encoding a polypeptide with amino acids 475 to515 of SEQ ID NO: 2.

[0032] According to another specific embodiment, the isolated orpurified nucleic acid molecule comprises a sequence encoding a humanHIF-3α polypeptide or degenerate variants thereof, the human HIF-3αpolypeptide or degenerate variant comprising part or all of amino acids475 to 515 of SEQ ID NO: 2. Preferably, the purified nucleic acidmolecule comprises part or all of nucleic acids 1423 to 1545 of SEQ IDNO: 1. In an even more specific aspect, the invention features anisolated or purified human nucleic acid molecule comprising apolynucleotide having the SEQ ID NO: 1, or degenerate variants thereof,and encoding a human HIF-3α polypeptide. Preferably, the nucleic acid isa cDNA and it encodes the amino acid sequence of SEQ ID NO: 2 or afragment thereof.

[0033] In a related aspect, the invention features an isolated orpurified nucleic acid molecule which hybridizes under low, preferablymoderate, and even more preferably high, stringency conditions to any ofthe nucleic acid molecules defined hereinbefore. More preferably, suchnucleic acid molecules hybridizes under moderate or high stringencyconditions with part or all of nucleic acids 1423 to 1545 of SEQ ID NO:1, or with part or all of a complementary sequence thereof.

[0034] The invention also features substantially pure human polypeptidesand proteins that are encoded by any of the above mentioned nucleicacids. In one embodiment, the protein has the biological activity of ahuman HIF-3α polypeptide. Preferred biological activity comprisesinduction of VEGF expression, thereby promoting angiogenesis.

[0035] In another embodiment, the invention aims at an isolated orpurified polypeptide comprising an amino acid sequence selected from thegroup consisting of:

[0036] a) sequences having at least 80% identity to SEQ ID NO: 2;

[0037] b) sequences having at least 85% homology to SEQ ID NO: 2;

[0038] c) sequence provided in SEQ ID NO: 2;

[0039] d) sequences having at least 80% identity to amino acid sequencesencoded by an open reading frame having SEQ ID NO: 3; and

[0040] e) sequences having at least 85% sequence homology to amino acidsequences encoded by an open reading frame having SEQ ID NO: 3.

[0041] More preferably, the polypeptide comprises an amino acid sequenceselected from the group consisting of:

[0042] a) sequences substantially the same as SEQ ID NO: 2; and

[0043] b) sequences substantially the same as amino acid sequencesencoded by an open reading frame having SEQ ID NO: 3.

[0044] In an even more specific aspect, the invention features asubstantially pure human HIF-3α polypeptide or a fragment thereof,comprising part or all of amino acids 475 to 515 of SEQ ID NO: 2, ordegenerate variants thereof. Even more preferably, the polypeptidecomprises an amino acid sequence 100% identical to SEQ ID NO: 2.

[0045] The present invention also features protein fragments derivedfrom any of the above mentioned protein or polypeptides. Similarly, theinvention further encompasses polypeptides fragment comprising an aminoacid sequence encoded by a nucleotide sequence comprising at least 24sequential nucleic acids of SEQ ID NO: 1 (hHIF-3α).

[0046] The present invention further features an antisense nucleic acidand a pharmaceutical composition comprising the same. Preferably, theantisense hybridizes under high stringency conditions to a genomicsequence or to a mRNA so that it reduces human HIF-3α cellular levels ofexpression. According to a first embodiment, the human HIF-3αpolypeptide comprises amino acids 475 to 515 of SEQ ID NO: 2. Morepreferably, the human HIF-3α polypeptide is encoded by an open readingframe having SEQ ID NO: 3. According to a specific embodiment, theantisense hybridizes under high stringency condition with part or all ofSEQ ID NO: 1, or with part or all of a complementary sequence thereof.More preferably, the antisense hybridizes under high stringencyconditions with part or all of nucleic acids 1423 to 1545 of SEQ ID NO:1, with part or all of nucleic acids 2116 to 2223 of SEQ ID NO: 1, orwith part or all of complementary sequences thereof.

[0047] The present invention further relates to a pharmaceuticalcomposition comprising: (1) at least one element selected from the groupconsisting of: (i) a nucleic acid molecule encoding a human HIF-3αpolypeptide; (ii) a human HIF-3α polypeptide; (iii) an antisense nucleicacid that reduces a human HIF-3α polypeptide levels of expression; and(iv) an isolated or purified antibody that specifically binds to aHIF-3α polypeptide; and (2) a pharmaceutically acceptable carrier ordiluent.

[0048] According to another aspect, the invention features a nucleotideprobe comprising a sequence of at least 15, 20, 25, 30, 40, 50, 75 or100 sequential nucleotides of SEQ ID NO: 1 or of a sequencecomplementary to SEQ ID NO: 1. Preferably, the probe comprises part orall of nucleic acids 1423 to 1545 of SEQ ID NO: 1, part or all ofnucleic acids 2116 to 2223 of SEQ ID NO: 1, part or all of nucleic acidsencoding amino acids 475 to 515 of SEQ ID NO: 2, or part or all of acomplementary sequence thereof. The invention also encompasses asubstantially pure nucleic acid that hybridizes under low, preferablymoderate, and more preferably under high stringency conditions to aprobe of at least 20, 25, 30, 40, 50, 75 or 100 nucleotides in lengththat is derived from SEQ ID NO: 1.

[0049] According to another aspect, the invention features a purifiedantibody. In a preferred embodiment, the antibody is a monoclonal or apolyclonal antibody that specifically binds to a purified mammalianHIF-3α polypeptide. Preferably, the antibody specifically binds to aHIF-3α polypeptide substantially the same as SEQ ID NO: 2. Morepreferably, the antibody specifically binds to a HIF-3α polypeptidecomprising part or all of amino acids 475 to 515 of SEQ ID NO: 2 andeven more preferably, the antibody specifically binds to part or all ofamino acids 475 to 515 of SEQ ID NO: 2.

[0050] In another aspect, the present invention further features amethod for inducing VEGF expression in a mammalian cell. The methodcomprises introducing and expressing in the cell a nucleic acid sequenceencoding polypeptide having the biological activity of a human HIF-3αpolypeptide. In a preferred embodiment, the cell consists of a cardiaccell located in the heart of a living mammal, and expression of thepolypeptide induces angiogenesis in cardiac tissue of the mammal. Inanother embodiment, the cell consists of a muscular cell located inmuscular tissue of a living mammal, and expression of the polypeptideinduces angiogenesis in the muscular tissue of the mammal. In anotherembodiment, the mammalian cell is a skeletal muscular cell therebyproviding a HIF-3α expressing-skeletal muscular cell, and the methodfurther comprises the step of transplanting a plurality of the HIF-3αexpressing-skeletal muscular cells in a cardiac tissue of a compatiblemammalian recipient.

[0051] Furthermore, the present invention features a method for inducingangiogenesis in a mammalian tissue having a plurality of cells, themethod comprising the step of introducing and expressing in at leastsome of these cells a nucleic acid sequence encoding a polypeptidehaving the biological activity of a human HIF-3α polypeptide.

[0052] The present invention also features a method for modulatingtumoral cell survival or for eliminating a tumoral cell in a mammal,comprising the step of reducing cellular expression levels of a HIF-3αpolypeptide. According to a preferred embodiment, the mammal consists ofa human, and a human HIF-3α antisense is introduced into the tumoralcell.

[0053] The present invention further features a method for determiningthe amount of a human HIF-3α polypeptide or a human HIF-3α nucleic acidin a biological sample, comprising the step of contacting the samplewith an antibody or with a probe as defined previously.

[0054] According to a further aspect, the invention features a method ofevaluating malignancy of a tumor in a human subject, comprising the stepof measuring the amount of a HIF-3α polypeptide or of a HIF-3α nucleicacid in a tumoral cell from the subject, the amount being indicative ofa degree of malignancy for the tumor.

[0055] In another related aspect, the invention features a kit fordetermining the amount of a HIF-3α polypeptide in a sample, the kitcomprising an antibody or a probe as defined previously, and at leastone element selected from the group consisting of instructions for usingthe kit, reaction buffer(s), and enzyme(s).

[0056] The nucleic acids of the invention may be incorporated into avector and or a cell (such as a mammalian, yeast, nematode or bacterialcell). The nucleic acids may also be incorporated into a transgenicanimal or embryo thereof. Therefore, the present invention featurescloning or expression vectors, and hosts (such as transformed ortransfected cells, transgenic animals) that contain any of the nucleicacids of the invention and more particularly those encoding a HIF-3αprotein, polypeptide or fragment, and those capable of directingexpression of a HIF-3α protein, polypeptide or fragment in avector-containing cell.

[0057] In a related aspect, the invention features a method forproducing a human HIF-3α polypeptide comprising:

[0058] providing a cell transformed with a nucleic acid sequenceencoding a human HIF-3α polypeptide positioned for expression in thiscell;

[0059] culturing the transformed cell under conditions suitable forexpressing the nucleic acid; and

[0060] producing the hHIF-3α polypeptide.

[0061] One of the greatest advantages of the present invention is thatit provides nucleic acid molecules, proteins, polypeptides, antibodies,probes, and cells that can be used for characterizing HIF-3α, modulateits cellular levels, and promotes angiogenesis.

[0062] Other objects and advantages of the present invention will beapparent upon reading the following non-restrictive description of thepreferred embodiments thereof and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063]FIG. 1 is a schema illustrating the human HIF-3α geneorganization.

[0064]FIG. 2A is a bar graph illustrating the expression of VEGF inHEK293 cells transfected with HIF-3α constructs.

[0065]FIG. 2B is a bar graph illustrating the expression of VEGF inHep3B cells transfected with HIF-3α constructs

[0066]FIG. 3A is a bar graph illustrating the expression of VEGF inHEK293 cells transfected with HIF-2α constructs.

[0067]FIG. 3B is a bar graph illustrating the expression of VEGF inHep3B cells transfected with HIF-2α constructs.

[0068]FIG. 3C is a bar graph illustrating the expression of VEGF inhuman skeletal muscle cells transduced with HIF-2α constructs.

[0069]FIG. 4 is a schema showing a possible process of angiogenesis andthe probable role therein of some angiogenesis-related genes.

[0070]FIG. 5A is a photograph of HIF-2α modified hSkMC implantedsubcutaneously in mice showing differentiation in myotubes and anorganized vasculature around and in between muscle fiber (arrows pointto vessels).

[0071]FIG. 5B is a bar graph that shows the angiogenic activity of HIFconstructs modified hSkMC.

[0072]FIG. 6A is a graphic showing the metabolic activity in an ischemicrat heart muscle treated with Ad.HIF-2α.

[0073]FIG. 6B is a bar graph that shows the measured blood vesseldensity in the infarcted area treated with myocardial HIF-2α genetransfer in rats.

DETAILED DESCRIPTION OF THE INVENTION

[0074] A) Definitions

[0075] Throughout the text, the word “kilobase” is generally abbreviatedas “kb”, the words “deoxyribonucleic acid” as “DNA”, the words“ribonucleic acid” as “RNA”, the words “complementary DNA” as “cDNA”,the words “polymerase chain reaction” as “PCR”, and the words “reversetranscription” as “RT”. Nucleotide sequences are written in the 5′ to 3′orientation unless stated otherwise.

[0076] In order to provide an even clearer and more consistentunderstanding of the specification and the claims, including the scopegiven herein to such terms, the following definitions are provided:

[0077] Antisense: as used herein in reference to nucleic acids, is meanta nucleic acid sequence, regardless of length, that is complementary tothe coding strand of a gene.

[0078] Expression: refers to the process by which gene encodedinformation is converted into the structures present and operating inthe cell. In the case of cDNAs, cDNA fragments and genomic DNAfragments, the transcribed nucleic acid is subsequently translated intoa peptide or a protein in order to carry out its function if any. By“positioned for expression” is meant that the DNA molecule is positionedadjacent to a DNA sequence which directs transcription and translationof the sequence (i.e., facilitates the production of, e.g., a HIF-3αpolypeptide, a recombinant protein or a RNA molecule).

[0079] Fragment: Refers to a section of a molecule, such as a protein, apolypeptide or a nucleic acid, and is meant to refer to any portion ofthe amino acid or nucleotide sequence.

[0080] Host: A cell, tissue, organ or organism capable of providingcellular components for allowing the expression of an exogenous nucleicacid embedded into a vector or a viral genome, and for allowing theproduction of viral particles encoded by such vector or viral genome.This term is intended to also include hosts which have been modified inorder to accomplish these functions. Bacteria, fungi, animal (cells,tissues, or organisms) and plant (cells, tissues, or organisms) areexamples of a host.

[0081] Isolated or Purified or Substantially pure: Means altered “by thehand of man” from its natural state, i.e., if it occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a protein/peptide naturally present in aliving organism is not “isolated”, the same polynucleotide separatedfrom the coexisting materials of its natural state, obtained by cloning,amplification and/or chemical synthesis is “isolated” as the term isemployed herein. Moreover, a polynucleotide or a protein/peptide that isintroduced into an organism by transformation, genetic manipulation orby any other recombinant method is “isolated” even if it is stillpresent in said organism.

[0082] Nucleic acid: Any DNA, RNA sequence or molecule having onenucleotide or more, including nucleotide sequences encoding a completegene. The term is intended to encompass all nucleic acids whetheroccurring naturally or non-naturally in a particular cell, tissue ororganism. This includes DNA and fragments thereof, RNA and fragmentsthereof, cDNAs and fragments thereof, expressed sequence tags,artificial sequences including randomized artificial sequences.

[0083] Open reading frame (“ORF”): The portion of a cDNA that istranslated into a protein. Typically, an open reading frame starts withan initiator ATG codon and ends with a termination codon (TAA, TAG orTGA).

[0084] Polypeptide: means any chain of more than two amino acids,regardless of post-translational modification such as glycosylation orphosphorylation.

[0085] Percent identity and Percent similarity: used herein incomparisons or nucleic acid and/or among amino acid sequences. Sequenceidentity is typically measured using sequence analysis software with thedefault parameters specified therein (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Owl 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

[0086] HIF-3α nucleic acid: means any nucleic acid (see above) encodinga mammalian polypeptide that has the biological activity of activating,in an hypoxia inducible fashion, target genes such as VEGF and having atleast 75%, 77%, 80%, 85%, 90%, 95%, 97% or 100% identity or homology tothe amino acid sequence shown in SEQ. ID. NO: 2. Even more preferably,the mammalian HIF-3α polypeptide comprises amino acids having at least75%, 77%, 80%, 85%, 90%, 95%, 97% or 100% identity or homology to aminoacids 475 to 515 of SEQ ID NO: 2. When referring to a human HIF-3αnucleic acid, the nucleic acid encoding SEQ. ID. NO: 2 is moreparticularly concerned. HIF-3α protein or HIF-3α polypeptide: means aprotein, a polypeptide, or a fragment thereof, encoded by a HIF3anucleic acid as described above.

[0087] Specifically binds: means an antibody that recognizes and binds aprotein or polypeptide but that does not substantially recognize andbind other molecules in a sample, e.g., a biological sample, thatnaturally includes protein.

[0088] Substantially the same: refers to nucleic acid or amino acidsequences having sequence variation that do not materially affect thenature of the protein. With particular reference to nucleic acidsequences, the term “substantially the same” is intended to refer to thecoding region and to conserved sequences governing expression, andrefers primarily to degenerate codons encoding the same amino acid, oralternate codons encoding conservative substitute amino acids in theencoded polypeptide. With reference to amino acid sequences, the term“substantially the same” refers generally to conservative substitutionsand/or variations in regions of the polypeptide not involved indetermination of structure or function of the protein.

[0089] Substantially pure polypeptide: means a polypeptide that has beenseparated from the components that naturally accompany it. Typically,the polypeptide is substantially pure when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the polypeptide is aHIF-3α polypeptide that is at least 75%, 80%, or 85%, more preferably atleast 90%, 95% or 97% and most preferably at least 99%, by weight, pure.A substantially pure HIF-3α polypeptide may be obtained, for example, byextraction from a natural source (including but not limited to lungcells, kidney cells, heart cells or any other cell expressing HIF-3α) byexpression of a recombinant nucleic acid encoding a HIF-3α polypeptide,or by chemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein is substantially free ofnaturally associated components when it is separated from thosecontaminants which accompany it in its natural state. Thus, a proteinwhich is chemically synthesized or produced in a cellular systemdifferent from the cell from which it naturally originates will besubstantially free from its naturally associated components.Accordingly, substantially pure polypeptides include those derived fromeukaryotic organisms but synthesized in E. coli or other prokaryotes. By“substantially pure DNA” is meant DNA that is free of the genes which,in the naturally-occurring genome of the organism from which the DNA ofthe invention is derived, flank the gene. The term therefore includes,for example, a recombinant DNA which is incorporated into a vector; intoan autonomously replicating plasmid or virus; or into the genomic DNA ofa prokaryote or eukaryote; or which exists as a separate molecule (e.g.,a cDNA or a genomic or cDNA fragment produced by PCR or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding an additionalpolypeptide sequence.

[0090] Transformed or Transfected or Transduced or Transgenic cell:refers to a cell into which (or into an ancestor of which) has beenintroduced, by means of recombinant DNA techniques, a DNA moleculeencoding (as used herein) a HIF-3α polypeptide. By “transformation” ismeant any method for introducing foreign molecules into a cell.Lipofection, calcium phosphate precipitation, retroviral delivery,electroporation, and ballistic transformation are just a few of theteachings which may be used.

[0091] Transgenic animal: any animal having a cell which includes a DNAsequence which has been inserted by artifice into the cell and becomespart of the genome of the animal which develops from that cell. As usedherein, the transgenic animals are usually mammalian (e.g., rodents suchas rats or mice) and the DNA (transgene) is inserted by artifice intothe nuclear genome.

[0092] Vector: A self-replicating RNA or DNA molecule which can be usedto transfer an RNA or DNA segment from one organism to another. Vectorsare particularly useful for manipulating genetic constructs anddifferent vectors may have properties particularly appropriate toexpress protein(s) in a recipient during cloning procedures and maycomprise different selectable markers. Bacterial plasmids are commonlyused vectors. Modified viruses such as adenoviruses and retroviruses areother examples of vectors.

[0093] B) General Overview of the Invention

[0094] The invention generally concerns a protein novel member of theHypoxia inducible factors (HIFs) HIF-3α. The present inventors have alsodiscovered uses for human HIF-3α proteins, fragments, nucleic acids, andantibodies for modulating HIF-3α cellular levels, for inducing VEGFexpression in a mammalian cell, and for inducing angiogenesis in amammalian tissue. This aspect of the invention also concerns the uses ofhuman HIF-3α proteins, fragments, nucleic acids, and antibodies formodulating HIF-3α cellular levels and for the treatment of coronary andcardiac diseases in mammals, including humans.

[0095] The invention also concerns methods and cells, and moreparticularly genetically modified muscular cells expressing a pluralityof angiogenesis-related genes, for inducing angiogenesis and forimproving muscular functions. This additional aspect of the invention isbased on the use of a nucleotide sequence encoding a transcriptionfactor from the hypoxia inducible factors family (HIF-1α, HIF-2α, andHIF-3α) and provides numerous advantages in the treatment of coronaryand cardiac diseases in mammals, including humans.

[0096] i) Cloning and Molecular Characterization of HIF-3α

[0097] As it will be described hereinafter in the exemplificationsection of the invention, the inventors have discovered, cloned andsequenced a human cDNA encoding a new human protein member from theHypoxia Inducible Factor family, called human HIF-3α (hHIF-3α).

[0098] The sequence of the HIF-3α cDNA and predicted amino acid sequenceis shown in the “Sequence Listing” section. SEQ ID NO: 1 corresponds tothe human HIF-3α cDNA and SEQ ID NO: 2 corresponds to the predictedamino acid sequence of the human protein. SEQ ID NO: 3 corresponds toHIF-3α Open reading frame.

[0099] The HIF-3α gene encodes a protein of 705 amino acids (A.A.) long.In silico analysis indicates that human HIF-3α protein has the followingfeatures: it has a molecular weight of about 76 kDa, an isoelectricpoint of about 5.95; an instability index of about 55.6 (i.e. unstable);an aliphatic index of about 80.6; and a grand average of hydropathicity(GRAVY) of about 0.388. It further comprises many potentialphosphorylation sites (45 Ser, 12 Thr, and 3 Tyr) and also manypotential phosphorylation sites. Predicted protein domains include theHelix Loop Helix (HLH) heterodimerization domain encoded by amino acids14-62. This domain is characteristic of HLH transcription factor family.Then, 2 PAS (A.A. 84-143 and 235-289) and 1 PAC (A.A. 295-337) domainsare identified, these domains are all common to the PAS family (asub-family to the HLH factors).

[0100] ii) HIF-3α Homology with Other Genes and Proteins

[0101] The cloning of hHIF-3α was carried out starting with the mouseHIF-3α sequence.

[0102] A blast search was made to identify sequence identity betweenhHIF-3α of the present invention, mHIF-3α and other existing sequences(see Table 1 hereinbelow). It was found that further to mice (GenBank™accession No. AF060194), HIF-3α had also been sequenced in rat (GenBank™accession No. NM_(—)022528).

[0103] Furthermore, the present inventors also found that their hHIF-3αsequence also shared high level of identity with another, recentlycloned hHIF-3α sequence (GenBank™ accession No. AB054067) published byHara et al. (Biochem. Biophys. Res. Comm. (2001), October 5;287:808-813). This sequence was published subsequent to the filing dateof the application on which the present application claims the benefit.The Hara et al. hHIF-3α sequence seems to be another isoform of HIF-3α,that differs from the hHIF-3α according to the present invention sinceit is depleted from nucleic acids 1423 to 1545 of SEQ ID NO: 1 encodingamino acids 475 to 515 of SEQ ID NO: 2. Equivalent mouse and ratsequences also lack amino acids homologues to 475-515 amino acids 475 to515 of SEQ ID NO: 2 and are thus homologues of Hara et al. hHIF-3αsequence. Although not shown, nucleic acids 7 to 345 of SEQ ID NO: 1also shares 100% identity with nucleotides 7 to 345 of a partial cDNAsequence of human HIF-3α published in 1999 (GenBank™ accession No.AF079154), this sequence encoding a 115 amino acid residues (GenBank™accession No. AAC99397), the last 113 amino acids sharing 100% identitywith amino acids 3 to 115 of SEQ ID NO: 2. TABLE 1 Identified isoformSequence identity and homology of human HIF-3α (SEQ ID NOs 1 and 2) toknown sequence Amino acid Amino acid Gene cDNA identity¹ identitysimilarity Human HIF-3α² 89% 93% 93% Mouse HIF-3α³ 74% 77% 82% RatHIF-3α⁴ 73% 75% 80%

[0104] Therefore, the present invention concerns an isolated or purifiednucleic acid molecule (such as cDNA) comprising a sequence selected fromthe group consisting of:

[0105] a) sequences provided in SEQ ID NO: 1 or 3;

[0106] b) complements of the sequences provided in SEQ ID NO: 1 or 3;

[0107] c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NO: 1 or 3;

[0108] d) sequences that hybridize to a sequence provided in SEQ ID NO:1 or 3, under moderately or strong stringent conditions;

[0109] e) sequences having at least 75% identity to a sequence of SEQ IDNO: 1 or 3; and

[0110] f) degenerate variants of a sequence provided in SEQ ID NO: 1 or3.

[0111] More preferably, the nucleic acid molecule of the inventioncomprises a sequence selected from the group consisting of:

[0112] a) a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%or 97% nucleotide sequence identity with SEQ ID NO: 1; and

[0113] b) a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%or 97% nucleotide sequence identity with a nucleic acid encoding anamino acid sequence of SEQ ID NO: 2.

[0114] More preferably, the nucleic acid molecule comprises a sequencesubstantially the same or having 100% identity with SEQ ID NO: 1 or asequence substantially the same or having 100% identity with nucleicacids encoding an amino acid sequence of SEQ ID NO: 2. Most preferrednucleic acid molecules are those comprising part or all of nucleic acids1423 to 1545 of SEQ ID NO: 1, and/or those comprising part or all ofnucleic acids encoding a polypeptide with amino acids 475 to 515 of SEQID NO: 2.

[0115] The present invention also concerns isolated or purified nucleicacid molecules comprising a sequence encoding a human HIF-3α polypeptideor degenerate variants thereof (the human HIF-3α polypeptide ordegenerate variant comprising part or all of amino acids 475 to 515 ofSEQ ID NO: 2) and purified nucleic acid molecules comprising part or allof nucleic acids 1423 to 1545 of SEQ ID NO: 1.

[0116] The present invention also concerns isolated or purified nucleicacid molecule which hybridizes under moderate, preferably highstringency conditions with part or all of any of the HIF-3α nucleic acidmolecules of the invention mentioned hereinbefore or with part or all ofa complementary sequence thereof. More preferably, the “hybridizing”nucleic acid hybridizes under moderate, preferably high stringencyconditions with part or all of nucleic acids 1423 to 1545 of SEQ ID NO:1, or with part or all of a complementary sequence thereof. The“hybridizing” nucleic acid could be used as probe or as antisensemolecules as it will be described hereinafter.

[0117] In a related aspect, the present invention concerns an isolatedor purified polypeptide, comprising an amino acid sequence selected fromthe group consisting of:

[0118] a) sequences encoded by part or all of the HIF-3α nucleic acidmolecules of the invention mentioned hereinbefore;

[0119] b) sequences having at least 80% identity to SEQ ID NO: 2;

[0120] c) sequences having at least 85% homology to SEQ ID NO: 2;

[0121] d) sequence provided in SEQ ID NO: 2;

[0122] e) sequences having at least 80% identity to amino acid sequencesencoded by an open reading frame having SEQ ID NO: 3; and

[0123] f) sequences having at least 85% sequence homology to amino acidsequences encoded by an open reading frame having SEQ ID NO: 3.

[0124] More preferably, the polypeptide comprises an amino acid sequencesubstantially the same or having 100% identity with SEQ ID NO: 2 or asequence substantially the same or having 100% identity with amino acidsequences encoded by an open reading frame having SEQ ID NO: 3. Eventmore preferably, the polypeptides comprise part or all of amino acids475 to 515 of SEQ ID NO: 2, or degenerate variants thereof, or comprisepart or all of amino acids encoded by nucleic acids 1423 to 1545 of SEQID NO: 1. Most preferred polypeptides are those having the biologicalactivity of a human HIF-3α polypeptide, such as VEGF expressioninducement capabilities for the promotion of angiogenesis.

[0125] iii) Vectors, Cells and Transgenic Animals

[0126] The invention is also directed to a host, such as a geneticallymodified cell, expressing a functional HIF-3α transcription factor.Preferably, the cell is a skeletal muscular cell or a cardiac cell.Preferably also, the cell comprises a cDNA encoding the transcriptionfactor.

[0127] The HIF-3α expressing cell may be a transiently-transfectedmammalian cell line (such as HEK293 cells, a Hep3B cells, and the like)or any suitable isolated primary cells, including by not limited tomammalian skeletal muscular cells, cardiac cells, bone marrow cells,fibroblasts, smooth muscle cells, endothelial cells, endothelialprogenitor cells and embryonic stem cells

[0128] A number of vectors suitable for stable transfection of mammaliancells are available to the public (e.g. plasmids, adenoviruses,adeno-associated viruses, retroviruses, Herpes Simplex Viruses,Alphaviruses, Lentiviruses), as are methods for constructing such celllines. The present invention encompasses any type of vector with aHIF-3α sequence.

[0129] The cells of the invention may be particularly useful whentransplanted in a compatible recipient for inducing angiogenesis,relieving ischemia, increasing the metabolic activity of a mammalianmuscular tissue, and/or increasing muscular function in CHF or inperipheral vascular disease, locally or in surrounding transplantedtissue. Of course, the genetically modified cells of the presentinvention could also be used for the formation of artificial organs orfor tissue constructions. HIF-3α expressing cells may also be used forproducing HIF-3α and derivatives thereof (see hereinafter).

[0130] The mammalian HIF-3α according to the present invention or afragment thereof may also be used to generate 1) transgenic animals thatexpress the HIF-3α gene or HIF-3α mutants at various levels in one ormultiple cell lineages, 2) knock-out animal in which expression of theendogenous HIF-3α gene is either prevented or regulated in one ormultiple cell lineages.

[0131] Characterization of HIF-3α genes provides information that isnecessary for a HIF-3α knockout animal model to be developed byhomologous recombination. Preferably, the model is a mammalian animal,most preferably a mouse. Similarly, an animal model of HIF-3αoverproduction may be generated by integrating one or more HIF-3αsequences into the genome, according to standard transgenic techniques.

[0132] iv) Synthesis of HIF-3α and Functional Derivative thereof

[0133] Knowledge of human HIF-3α gene sequence open the door to a seriesof applications. For instance, the characteristics of the cloned HIF-3αgene sequence may be analyzed by introducing the sequence into variouscell types or using in vitro extracellular systems. The function ofHIF-3α may then be examined under different physiological conditions.The HIF-3α cDNA sequence may be manipulated in studies to understand theexpression of the gene and gene product. Alternatively, cell lines maybe produced which overexpress the gene product allowing purification ofHIF-3α for biochemical characterization, large-scale production,antibody production, and patient therapy.

[0134] For protein expression, eukaryotic and prokaryotic expressionsystems may be generated in which the HIF-3α gene sequence is introducedinto a plasmid or other vector which is then introduced into livingcells. Constructs in which the HIF-3α cDNA sequence containing theentire open reading frame inserted in the correct orientation into anexpression plasmid may be used for protein expression. Alternatively,portions of the sequence, including wild-type or mutant HIF-3αsequences, may be inserted. Prokaryotic and eukaryotic expressionsystems allow various important functional domains of the protein to berecovered as fusion proteins and then used for binding, structural andfunctional studies and also for the generation of appropriateantibodies.

[0135] Eukaryotic expression systems permit appropriatepost-translational modifications to expressed proteins. This allows forstudies of the HIF-3α gene and gene product including determination ofproper expression and post-translational modifications for biologicalactivity, identifying regulatory elements located in the 5′ region ofthe HIF-3α gene and their role in tissue regulation of proteinexpression. It also permits the production of large amounts of normaland mutant proteins for isolation and purification, to use cellsexpressing HIF-3α as a functional assay system for antibodies generatedagainst the protein, to test the effectiveness of pharmacological agentsor as a component of a signal transduction system, to study the functionof the normal complete protein, specific portions of the protein, or ofnaturally occurring polymorphisms and artificially produced mutatedproteins. The HIF-3α DNA sequence may be altered by using proceduressuch as restriction enzyme digestion, DNA polymerase fill-in,exonuclease deletion, terminal deoxynucleotide transferase extension,ligation of synthetic or cloned DNA sequences and site directed sequencealteration using specific oligonucleotides together with PCR.

[0136] Accordingly, the invention also concerns a method for producing ahuman a human HIF-3α polypeptide. The method comprises the steps of: (i)providing a cell transformed with a nucleic acid sequence encoding ahuman HIF-3α polypeptide positioned for expression in the cell; (ii)culturing the transformed cell under conditions suitable for expressingthe nucleic acid; (iii) producing said a human HIF-3α polypeptide; andoptionally, (iv) recovering the human HIF-3α polypeptide produced.

[0137] Once the recombinant protein is expressed, it is isolated by, forexample, affinity chromatography. In one example, an anti-HIF-3αantibody, which may be produced by the methods described herein, can beattached to a column and used to isolate the HIF-3α protein. Lysis andfractionation of HIF-3α-harboring cells prior to affinity chromatographymay be performed by standard methods. Once isolated, the recombinantprotein can, if desired, be purified further.

[0138] Methods and techniques for expressing recombinant proteins andforeign sequences in prokaryotes and eukaryotes are well known in theart and will not be described in more detail. One can refer, ifnecessary to Joseph Sambrook, David W. Russell, Joe Sambrook MolecularCloning: A Laboratory Manual 2001 Cold Spring Harbor Laboratory Press.Those skilled in the art of molecular biology will understand that awide variety of expression systems may be used to produce therecombinant protein. The precise host cell used is not critical to theinvention. The HIF-3α protein may be produced in a prokaryotic host(e.g., E. coil) or in a eukaryotic host (e.g., S. cerevisiae, insectcells such as Sf21 cells, or mammalian cells such as COS-1, NIH 3T3, orHeLa cells). These cells are publicly available, for example, from theAmerican Type Culture Collection, Rockville, Md. The method oftransduction and the choice of expression vehicle will depend on thehost system selected.

[0139] Polypeptides of the invention, particularly short HIF-3αfragments, may also be produced by chemical synthesis. These generaltechniques of polypeptide expression and purification can also be usedto produce and isolate useful HIF-3α fragments or analogs, as describedherein.

[0140] Skilled artisans will recognize that a mammalian HIF-3α, or afragment thereof (as described herein), may serve as an activeingredient in a therapeutic composition. This composition, depending onthe HIF-3α or fragment included, may be used to regulate cellproliferation, survival and angiogenesis and thereby treat any conditionthat is caused by a disturbance in cell proliferation, accumulation orreplacement. Thus, it will be understood that another aspect of theinvention described herein, includes the compounds of the invention in apharmaceutically acceptable carrier.

[0141] v) Upregulation of HIF-3α Expression for Promoting Angiogenesis

[0142] Knowledge of human HIF-3α gene sequence provides novel promisingapproaches for patient therapy. As it will be shown in detailshereinafter in the exemplification section of the application,upregulation of HIF-3α expression could be used to increase VEGFexpression in mammalian cells and thereby promote angiogenesis inischemic and non-ischemic tissue of mammals, preferably animal modelsand humans.

[0143] Therefore, the invention also relates to methods for inducingVEGF expression in a mammalian cell by introducing and expressing in thecell a nucleic acid sequence encoding polypeptide having the biologicalactivity of a human HIF-3α polypeptide. Of course, other nucleic acids,such as those which expression is known to also induce VEGF expression,may be introduced and expressed in the cell together with HIF-3α.

[0144] Another related aspect of the invention concerns methods forinducing angiogenesis in a mammalian tissue having a plurality of cells,by introducing and expressing in at least some of these cells a nucleicacid sequence encoding a polypeptide having the biological activity of ahuman HIF-3α polypeptide.

[0145] Preferably, these methods are achieved by transfecting in vitro,in vivo or ex vivo cells with a HIF-3α cDNA, the human HIF-3αpolypeptide comprises an amino acid sequence substantially the same asSEQ ID NO: 2.

[0146] As mentioned previously in the “background” section, stimulationof angiogenesis may be beneficial for the treatment of coronary heartdiseases. Therefore, the human HIF-3α sequence of the invention could beadvantageously used for such purposes. In one embodiment, the cellconsists of a cardiac cell located in the heart of a living mammal, andthe HIF-3α nucleic acid sequence is introduced in a plurality of thesecardiac cell such that expression of the HIF-3α polypeptide induceangiogenesis in cardiac tissue of the mammal. Introduction of the HIF-3αnucleic acid sequence may be done by using a vector as definedpreviously or by any suitable technique known in the art.

[0147] In another embodiment, the cell consists of an isolated muscularcell (preferably skeletal muscular cell), and this cell is geneticallymodified so as to express the HIF-3α polypeptide. Genetically modifiedHIF-3α expressing cells are then transplanted in tissue (e.g. cardiactissue) of a compatible mammalian recipient. More preferably, thetransplantation is autologous (the cells are isolated from musculartissue, such as leg, of the recipient), and the cells are transplantedto the recipient (such as an injection in the scar of the heart) in anamount that is sufficient to induce angiogenesis locally or insurrounding transplanted tissue.

[0148] In yet another embodiment, the cell consists of a muscular celllocated in muscular tissue of a living mammal, and expression of theHIF-3α polypeptide induce angiogenesis in the muscular tissue of themammal (autologous or heterologous transplantation). This method isparticularly useful for treating peripheral artery diseases (e.g.ischemia in the legs due to femoral or upstream artery obstruction inhumans).

[0149] The nucleotide sequence may be introduced in the cell or tissueusing well known methods. Indeed, the sequence(s) may be introduceddirectly in the cells of a given tissue, injected in the tissue, orintroduced via the transplantation of previously genetically modifiedcompatible cells (see hereinafter). Methods for introducing a nucleotidesequence into eukaryote cells such as mammalian muscular cells or forgenetically modifying such cells are well known in the art. Forinstance, this may be achieved with adenoviral vectors, plasmid DNAtransfer (naked DNA or complexed with liposomes) or electroporation. Ifnecessary, a person skilled in the art may look at Isner J., (Nature(2002), 415:234-239) for a review of myocardial gene therapy methods andto U.S. patent application US20010041679A1 or U.S. Pat. No. 5,792,453which provides methods of gene transfer-mediated angiogenesis therapy.Preferably, the level of expression of the transcription factor(s) issuch that the angiogenesis-related gene is expressed at a level that issufficient to induce angiogenesis locally or in surrounding tissue. Forbetter controlling its expression and selectivity, the transcriptionfactor may be inducible.

[0150] In preferred embodiments, a plurality of genetically modifiedskeletal muscular cells are transplanted into the heart of a compatiblerecipient. Preferably, the transplantation is autologous. Morepreferably, the cells are transplanted in an amount that is sufficientto induce angiogenesis locally or in surrounding transplanted tissue.Even more preferably, the transplantation improves the recipient'scardiac functions. Transplantation methods, are well known in the art.For detailed examples of muscular cell transplantation, one may refer toU.S. Pat. Nos. 5,602,301 and 6,099,832.

[0151] vi) Downregulation of HIF-3α Expression

[0152] As mentioned previously, HIF-3α expression induces VEGFexpression, which itself promotes angiogenesis. Since it is well knownthat tumoral cell survival depend on angiogenesis, we propose that, insome tumors, HIF-3α expression is essential for cancer cellproliferation. Accordingly, downmodulation of HIF-3α could be used toprevent and/or treat these tumors.

[0153] Therefore, the invention relates to methods for modulatingtumoral cell survival or for eliminating a tumoral cell in a human byreducing cellular expression levels of a human HIF-3α polypeptide. In apreferred embodiment, this is achieved by delivering an antisense intothe tumoral cells. This can be achieved by intravenous injection,intratumoral injection or other local drug delivery using currentlyavailable methods (e.g. Crooke et al., (2000), Oncogene 19, 6651-6659;Stein et al. (2001), J Clin. Invest 108, 641-644; and Tamm et al.,(2001), Lancet 358, 489-497).

[0154] According to a related aspect of the above-mentioned method, theinvention relates to antisense nucleic acids and to pharmaceuticalcompositions comprising such antisenses, the antisense being capable ofreducing HIF-3α cellular levels of expression, and more particularly thelevel of expression of human HIF-3α polypeptide encoded by an openreading frame having SEQ ID NO: 3 and/or comprising amino acids 475 to515 of SEQ ID NO: 2. Preferably, the antisense nucleic acid iscomplementary to a nucleic acid sequence encoding a hHIF-3α protein orencoding any of the polypeptides derived therefrom. More preferably, theantisense hybridizes under high stringency conditions to a genomicsequence or to a mRNA, even more preferably under high stringencyconditions with part or all of SEQ ID NO: 1, or with part or all of acomplementary sequence thereof. Most preferred antisense molecules arethose which hybridize under high stringency conditions with part or allof nucleic acids 1423 to 1545 of SEQ ID NO: 1, with part or all ofnucleic acids 2116 to 2223 of SEQ ID NO: 1, or with part or all ofcomplementary sequences thereof.

[0155] A non-limitative example of high stringency conditions includes:

[0156] a) pre-hybridization and hybridization at 68° C. in a solution of5×SSPE (1×SSPE=0.18 M NaCl, 10 mM NaH₂PO₄); 5×Denhardt solution; 0.05%(w/v) sodium dodecyl sulfate (SDS); et 100 μg/ml salmon sperm DNA;

[0157] b) two washings for 10 min at room temperature with 2×SSPE and0.1% SDS;

[0158] c) one washing at 60° C. for 15 min with 1×SSPE and 0.1% SDS; and

[0159] d) one washing at 60° C. for 15 min with 0.1×SSPE et 0.1% SDS.

[0160] vii) HIF-3α Antibodies

[0161] The invention features purified antibodies that specificallybinds to a HIF-3α protein. The antibodies of the invention may beprepared by a variety of methods using the HIF-3α proteins orpolypeptides described above. For example, the HIF-3α polypeptide, orantigenic fragments thereof, may be administered to an animal in orderto induce the production of polygonal antibodies. Alternatively,antibodies used as described herein may be monoclonal antibodies, whichare prepared using hybridoma technology (see, e.g., Hammerling et al.,In Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., 1981).The invention features antibodies that specifically bind human HIF-3αpolypeptides, or fragments thereof. In particular, the inventionfeatures “neutralizing” antibodies. By “neutralizing” antibodies ismeant antibodies that interfere with any of the biological activities ofthe HIF-3α polypeptide, particularly the ability of HIF-3α to induceVEGF expression. The neutralizing antibody may reduce the ability ofHIF-3α polypeptides to inhibit VEGF expression by, preferably 50%, morepreferably by 70%, and most preferably by 90% or more. Any standardassay of VEGF expression, including those described herein, may be usedto assess potentially neutralizing antibodies. Once produced, monoclonaland polyclonal antibodies are preferably tested for specific HIF-3αrecognition by Western blot, immunoprecipitation analysis or any othersuitable method.

[0162] In addition to intact monoclonal and polyclonal anti-HIF-3αantibodies, the invention features various genetically engineeredantibodies, humanized antibodies, and antibody fragments, includingF(ab′)2, Fab′, Fab, Fv and sFv fragments. Antibodies can be humanized bymethods known in the art. Fully human antibodies, such as thoseexpressed in transgenic animals, are also features of the invention.

[0163] Antibodies that specifically recognize HIF-3α (or fragments ofHIF-3α), such as those described herein, are considered useful to theinvention. Such an antibody may be used in any standard immunodetectionmethod for the detection, quantification, and purification of a HIF-3αpolypeptide. Preferably, the antibody binds specifically to HIF-3α. Theantibody may be a monoclonal or a polyclonal antibody and may bemodified for diagnostic or for therapeutic purposes. More preferably theantibody specifically binds the a HIF-3α polypeptide comprising part orall of amino acids 475 to 515 of SEQ ID NO: 2. The most preferredantibodies are those that specifically binds to part or all of aminoacids 475 to 515 of SEQ ID NO: 2.

[0164] The antibodies of the invention may, for example, be used in animmunoassay to monitor HIF-3α expression levels, to determine thesubcellular location of a HIF-3α or HIF-3α fragment produced by a mammalor to determine the amount of HIF-3α or fragment thereof in a biologicalsample. Antibodies that inhibit HIF-3α described herein may beespecially useful for conditions where decreased HIF-3α function wouldbe advantageous such as inhibition of cancer cell proliferation (seehereinafter). In addition, the antibodies may be coupled to compoundsfor diagnostic and/or therapeutic uses such as radionucleotides forimaging and therapy and liposomes for the targeting of compounds to aspecific tissue location. The antibodies may also be labeled (e.g.immunofluorescence) for easier detection.

[0165] viii) Administration of HIF-3α Polypeptides, Modulators of HIF-3αSynthesis or Function

[0166] Therapies may be designed to circumvent or overcome an inadequateHIF-3α gene expression. This could be accomplished for instance bytransfection of HIF-3α cDNA.

[0167] To obtain large amounts of pure HIF-3α, cultured cell systemswould be preferred. Delivery of the protein to the affected tissues canthen be accomplished using appropriate packaging or administratingsystems. Alternatively, it is conceivable that small molecule analogscould be used and administered to act as HIF-3α agonists and in thismanner produce a desired physiological effect. Methods for finding suchmolecules are provided herein.

[0168] A HIF-3α protein or polypeptide, polypeptide, antibody ormodulator (e.g. antisense) may be administered within a pharmaceuticallyacceptable diluent, carrier, or excipient, in unit dosage form.Conventional pharmaceutical practice may be used to provide suitableformulations or compositions to administer HIF-3α protein, polypeptide,or modulator to patients. Administration may begin before the patient issymptomatic. Any appropriate route of administration may be employed,for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration. Therapeutic formulations may be in the form ofliquid solutions or suspensions; for oral administration, formulationsmay be in the form of tablets or capsules; and for intranasalformulations, in the form of powders, nasal drops, or aerosols.

[0169] Methods well known in the art for making formulations are found,for example, in “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

[0170] If desired, treatment with a HIF-3α protein, polypeptide, ormodulatory compound may be combined with more traditional therapies forthe disease such as surgery, steroid therapy, or chemotherapy.

[0171] According to a preferred embodiment, a HIF-3α antisense would beincorporated in a pharmaceutical composition comprising at least one ofthe oligonucleotides defined previously, and a pharmaceuticallyacceptable carrier. The amount of antisense present in the compositionof the present invention is a therapeutically effective amount. Atherapeutically effective amount of antisense is that amount necessaryso that the antisense performs its biological function without causingoverly negative effects in the host to which the composition isadministered. The exact amount of oligonucleotides to be used andcomposition to be administered will vary according to factors such asthe oligo biological activity, the type of condition being treated, themode of administration, as well as the other ingredients in thecomposition. Typically, the composition will be composed of about 1% toabout 90% of antisense, and about 20 μg to about 20 mg of antisense willbe administered. For preparing and administering antisenses as well aspharmaceutical compositions comprising the same, methods well known inthe art may be used. For instance, see Crooke et al (Oncogene, 2000,19:6651-6659) and Tamm et al. (Lancet 200,1358:489-497) for a review ofantisense technology in cancer chemotherapy.

[0172] ix) Assessment of HIF-3α Intracellular or Extracellular Levels

[0173] As noted, the antibodies and probes described above may be usedto monitor HIF-3α protein expression and/or to determine the amount ofHIF-3α or fragment thereof in a biological sample, and/or to evaluatemalignancy of a tumor in a human subject.

[0174] In addition, in situ hybridization may be used to detect theexpression of the HIF-3α gene. As it is well known in the art, in situhybridization relies upon the hybridization of a specifically labelednucleic acid probe to the cellular RNA in individual cells or tissues.Therefore, oligonucleotides or cloned nucleotide (RNA or DNA) fragmentscorresponding to unique portions of the HIF-3α gene may be used to assesHIF-3α cellular levels or detect specific mRNA species. Such anassessment may also be done in vitro using well known methods (Northernanalysis, quantitative PCR, etc.).

[0175] Determination of the amount of HIF-3α or fragment thereof in abiological sample may be especially useful for diagnosing a cellproliferative disease or an increased likelihood of such a disease,particularly in a human subject, using a HIF-3α nucleic acid probe orHIF-3α antibody. The present inventor also suspect that there exist acorrelation between the degree of malignancy of certain types of tumorwith the amount HIF-3α or fragment thereof, and that high levels ofHIF-3α is indicative that the tumoral cells have a higher angiogenesisactivity and malignancy. Highly malignant cancers are cancers whichcells display a short doubling time (e.g. hematopoietic cancer, lungcancers, prostate cancer, testis cancer, breast cancer, melanomas,pancreatic cancer intestine cancers, sarcomas, prostate cancer andhematologic cancers).

[0176] The methods of the invention may be carried out by contacting, invitro or in vivo, a biological sample (such as a blood sample or atissue biopsy) from an individual suspected of harboring cancer cells,with a HIF-3α antibody or a probe according to the invention, in orderto evaluate the amount of HIF-3α in the sample or the cells therein. Themeasured amount would be indicative of the probability of the subject ofhaving proliferating tumoral cells since it is expected that these cellshave a higher level of HIF-3α expression.

[0177] In a related aspect, the invention features a method fordetecting the expression of HIF-3α in tissues comprising, i) providing atissue or cellular sample; ii) incubating said sample with ananti-HIF-3α polyclonal or monoclonal antibody; and iii) visualizing thedistribution of HIF-3α.

[0178] Assay kits for determining the amount of HIF-3α in a sample wouldalso be useful and are within the scope of the present invention. Such akit would preferably comprise HIF-3α antibody(ies) or probe(s) accordingto the invention and at least one element selected from the groupconsisting of instructions for using the kit, assay tubes, enzymes,reagents or reaction buffer(s), enzyme(s).

[0179] x) Identification of Molecules that Modulate HIF-3α ProteinExpression

[0180] HIF-3α cDNA may be used to facilitate the identification ofmolecules that increase or decrease HIF-3α expression. In one approach,candidate molecules are added, in varying concentration, to the culturemedium of cells expressing HIF-3α mRNA. HIF-3α expression is thenmeasured (or expression of another gene, such as VEGF which expressionis regulated by HIF-3α), for example, by Northern blot analysis using aHIF-3α cDNA, or cDNA or RNA fragment, as a hybridization probe. Thelevel of HIF-3α expression in the presence of the candidate molecule iscompared to the level of HIF-3α expression in the absence of thecandidate molecule, all other factors (e.g. cell type and cultureconditions) being equal.

[0181] Compounds that modulate the level of HIF-3α may be purified, orsubstantially purified, or may be one component of a mixture ofcompounds such as an extract or supernatant obtained from cells (Ausubelet al., supra). In an assay of a mixture of compounds, HIF-3α expressionis tested against progressively smaller subsets of the compound pool(e.g., produced by standard purification techniques such as HPLC orFPLC) until a single compound or minimal number of effective compoundsis demonstrated to modulate HIF-3α expression.

[0182] Compounds may also be screened for their ability to modulateHIF-3α-biological activity (e.g. VEGF expression, induction ofangiogenesis). In this approach, the biological activity of HIF-3α or ofa cell expressing HIF-3α (e.g. lung or kidney cell) in the presence of acandidate compound is compared to the biological activity in itsabsence, under equivalent conditions. Again, the screen may begin with apool of candidate compounds, from which one or more useful modulatorcompounds are isolated in a step-wise fashion. The HIF-3α or cellbiological activity may be measured by any suitable standard assay.

[0183] The effect of candidate molecules on HIF-3α-biological activitymay, instead, be measured at the level of translation by using thegeneral approach described above with standard protein detectiontechniques, such as Western blotting or immunoprecipitation with aHIF-3α-specific antibody (for example, the HIF-3α antibody describedherein).

[0184] Another method for detecting compounds that modulate the activityof HIF-3α is to screen for compounds that interact physically with agiven HIF-3α polypeptide. Depending on the nature of the compounds to betested, the binding interaction may be measured using methods such asenzyme-linked immunosorbent assays (ELISA), filter binding assays, FRETassays, scintillation proximity assays, microscopic visualization,immunostaining of the cells, in situ hybridization, PCR, etc.

[0185] A molecule that promotes an increase in HIF-3α expression orHIF-3α activity is considered particularly useful to the invention; sucha molecule may be used, for example, as a therapeutic to increasecellular levels of HIF-3α and thereby exploit the ability of HIF-3αpolypeptides to promote and/or induce angiogenesis.

[0186] A molecule that decreases HIF-3α activity (e.g., by decreasingHIF-3α gene expression or polypeptide activity) may be used to decreaseand/or block angiogenesis and/or cellular proliferation. This would beadvantageous in the treatment of cancer.

[0187] Molecules that are found, by the methods described above, toeffectively modulate HIF-3α gene expression or polypeptide activity, maybe tested further in animal models. If they continue to functionsuccessfully in an in vivo setting, they may be used as therapeutics toeither promotes or inhibit angiogenesis.

[0188] xi) Induction of Expression of Angiogenesis-related Gene(s) byHIF-2α and HIF-3α

[0189] According to a related aspect, the present invention relates tomethods and cells for inducing angiogenesis and for improving muscularfunction, and more particularly for treating coronary and cardiacdiseases in mammals. The invention also provides genetically modifiedmuscular cells expressing a plurality of angiogenesis-related genes.

[0190] This aspect of the invention is based on the use of a nucleotidesequence encoding a transcription factor from the hypoxia induciblefactors family (HIF-1α, HIF-2α, and HIF-3α). As it will be shown in theexemplification section, the present inventors have demonstrated thatHIF-2α was stimulating, in addition to VEGF, the expression of othermolecules implicated in angiogenesis such as IL-8, IL-6, PIGF, LIFreceptor, PAI-2 and MMP7 in muscular cells. The inventors showed, in amodel of rat CHF, that HIF-2α could be used for therapeutic angiogenesisin mammals. Also, the inventors have genetically modified skeletalmuscle cells (SkMC) with HIF-2α gene and showed that these cellsdemonstrated superior angiogenic properties in vivo. Furthermore,treatment of CHF rats with HIF-2α gene transfer resulted inangiogenesis, higher metabolic activity and improved cardiac functions.

[0191] Since HIF-1α, HIF-2α, and HIF-3α belong to the same family andare closely related, it is expected that similar results could beobtained with any of the HIF members. Therefore, the additional aspectsof the present invention given in the present section encompasses notonly the use of HIF-2α but HIF-1α and HIF-3α (all isoforms) as well.HIF-1α (GenBank™ accession No. U22431) is described in by Wang et al.,(Proc. Natl. Aca. Sci. USA (1995) 92: 5510-5514) and in U.S Pat. Nos.5,882,314; 6,020,462 and 6,124,131. HIF-2α (GenBank™ accession No.U81984) is described by Tian et al., (Genes & Dev. (1996) 11: 72-82),and in U.S. Pat. No. 5,692,963. One isoform of HIF-3α is describedherein and another isoform (GenBank™ accession No. AB054067) has beendescribed by Hara et al. (Biochem. Biophys. Res. Comm. (2001), October5; 287:808-813). All these documents are incorporated herein byreference.

[0192] Therefore, this aspect of the invention is directed to a methodfor inducing in a muscular mammalian cell the expression of at leastone, preferably a plurality of angiogenesis-related gene(s), the methodcomprising the step of introducing and expressing in the cell a nucleicacid sequence encoding a functional HIF-2α transcription factor or afunctional HIF-3α transcription factor.

[0193] According to another related aspect, the invention is directed toa method for increasing the metabolic activity of a muscular cell (suchas glucose consumption), comprising the step of introducing andexpressing in the cell a nucleic acid sequence encoding a functionaltranscription factor of the Hypoxia Inducible Factor (HIF) family.Preferably, the transcription factor is HIF-2α or HIF-3α.

[0194] In a further related aspect, the invention is directed to amethod for improving cardiac tissue functions of a mammal, comprisingthe step of providing to the cardiac tissue of the mammal a plurality ofgenetically modified cells expressing a nucleic acid sequence encoding afunctional HIF-2α transcription factor or a functional HIF-3αtranscription factor.

[0195] According to another related aspect, the invention is directed toa method for inducing angiogenesis in a mammalian muscular tissue,comprising the step of providing the muscular tissue with a plurality ofgenetically modified muscular cells expressing a nucleic acid sequenceencoding a functional HIF-2α transcription factor or a functional HIF-3αtranscription factor.

[0196] According to the invention, a nucleotide sequence encoding atranscription factor of hypoxia inducible factor family is introducedand expressed into a muscular cell, preferably a skeletal muscular cellor a cardiac cell by using any suitable method including but not limitedto adenoviral infection, and plasmid, cosmid or artificial chromosometransfection or electroporation. More preferably, the nucleic acidsequence encoding the transcription factor(s) is a cDNA.

[0197] In a further aspect, the invention is directed to a geneticallymodified muscular cell (e.g. skeletal muscle cell, cardiac cell)expressing a functional HIF-2α transcription factor or a functionalHIF-3α transcription factor. Preferably, the cell is a skeletal muscularcell or a cardiac cell. Preferably also, the cell comprises a cDNAencoding the transcription factor. As mentioned previously, such cellsmay be particularly useful when transplanted in a compatible recipientfor inducing angiogenesis, relieving ischemia, increasing the metabolicactivity of a mammalian muscular tissue, and/or increasing muscularfunction in CHF or in peripheral vascular disease, locally or insurrounding transplanted tissue. Of course, the genetically modifiedcells of the present invention could also be used for the formation ofartificial organs or for tissue constructions.

[0198] Although transplantation of cells (autologous transplantation orfrom a compatible donor) is preferred for inducing angiogenesis locally,in surrounding tissue and/or for improving the metabolic activity of amuscular cell and/or for relieving ischemia in coronary heart disease orin peripheral vascular disease and/or for improving the mammal's cardiacfunctions, the nucleic acid sequence encoding the functionaltranscription factor may be introduced directly in the tissue of themammal using any suitable method known in the art (see hereinbefore forsome examples). The angiogenesis-related gene should be expressed at alevel that is sufficient to induce angiogenesis locally or insurrounding tissue.

[0199] As it will now be demonstrated by way of an example hereinafter,the present invention is useful for inducing angiogenesis, relievingischemia and increasing cell metabolic activity and tissue function inCHD and in PVD.

EXAMPLES

[0200] The following examples are illustrative of the wide range ofapplicability of the present invention and is not intended to limit itsscope. Modifications and variations can be made therein withoutdeparting from the spirit and scope of the invention. Although anymethod and material similar or equivalent to those described herein canbe used in the practice for testing of the present invention, thepreferred methods and materials are described.

Example 1 Use of HIF-1α, HIF-2α and HIF-3α for Inducing Angiogenesis andImproving Muscular Functions

[0201] Materials and Methods

[0202] Plasmids Construction

[0203] HIF-1α-VP16/pcDNA3 was obtained from S. L. McKnight (Tian et al.,Genes & Dev. (1996) 11:72-82). To generate HIF-1α-VP16/pcDNA3, a VP16fragment (NheI and blunt ended EcoRI from pVP16 (ClonTech)) was insertedin HIF-1α/pcDNA3 cut with Af/II and XbaI blunt ended in presence of aNheI-Af/II linker composed of the oligos TTA AGA TAT CGA TGA CAC GTG(SEQ ID NO: 4) and TCA GCA CGT GTC ATC GAT ATC (SEQ ID NO: 5), replacingthe 3′ part of HIF-1α sequence by VP16, putting DNA binding domain andheterodimerization domains (HLH and PAS) of HIF-1α (encoded by the 5′ ofthe gene) in frame with VP16 transcription activation domain.

[0204] HIF-2α-VP16/pcDNA3 was obtained from S. L. McKnight (Tian et al.,Genes & Dev. (1996) 11:72-82). To generate HIF-2α-VP16/pcDNA3, a HIF-2α5′ region was amplified by PCR using HIF-2α/pcDNA3 as template with theoligonucleotides T7 and GCT AGC TAG GM GTT ACT CCT CTC (SEQ ID NO: 6),creating a NheI at the end of HIF-2α DNA binding domain andheterodimerization domains (HLH-PAS). This fragment was cut with NheIand KpnI and was inserted in place of HIF-1α sequence inHIF-1-VP16/pcDNA3 cut NheI and KpnI. Sequencing confirmed integrity ofthe amplified sequence.

[0205] HIF-3α was cloned by RT-PCR by homology deduction from mouseHIF-3α sequence. Using Marathon™ RT-PCR kit (ClonTech), 2 fragments ofHIF-3α sequence were amplified from adult human heart RNA (ClonTech)with the oligos CCA TGG ACA GGT CGA CCA CGG AGC TGC GCA AGG (SEQ ID NO:7) (containing an ATG initiator in a NcoI site) and CGC AGG CAG GTG GCTTGT AGG CCC T (SEQ ID NO: 8) for the 5′ end and with the oligos CAG CTGGAG CTC ATT GGA CAC AGC ATC (SEQ ID NO: 9) and CCC CAT CCT GTG CGT TGGCTG CCG (SEQ ID NO: 10) for the 3′ end. Both fragments were sequencedand put together with the unique Ndel site, and the reconstructed cDNAwas inserted in the NcoI initiator of HIF-1α/pcDNA3, using HIF-1α Kozaksequence to initiate translation. To generate HIF-3α-VP16/pcDNA3, a PCRfragment containing HIF-3α DNA binding domain and heterodimerizationdomains (HLH and PAS) was amplified using HIF-3α/pcDNA3 as template withthe oligos T7 and GGA GTC AGC TTA AGC TGA ATG GGT CTG C (SEQ ID NO: 11).The amplification product was cut with BamHI and Af/II (coming from the3′ oligo) and inserted in place of HIF-1α sequence in HIF-1α-VP16/pcDNA3cut with BamHI and Af/II. Sequencing confirmed integrity of theamplified sequence.

[0206] Transfection

[0207] Early passage 293 cells (ATCC # CRL-157) were plated at 1,7×10⁶cells per plate (100 mm) and grown overnight. 10 μg of sterile plasmidDNA was transfected with lipofectamin, according to the instructionsfrom the manufacturer. After a 5 hours transfection, cells wereincubated either in normoxia (5% CO₂ in normal air at 37° C.) or inhypoxia (5% CO₂, 2% O₂ at 37° C.) for 24 hours. ˜70% confluence Hep3Bcells (ATCC #HB-8064) in 100 mm dishes were transfected with 4 μg ofsterile plasmid DNA with 6 μl of Fugene 6™ (Roche), according to theinstructions from the manufacturer.

[0208] Adenovirus Production

[0209] HIF constructs were used to produce adenoviral vectors with theAd.Easy™ technology using manufacturer methodology (Q-Biogene).

[0210] Infection

[0211] Early passage human SkMC (Cambrex #CC-2561) were plated in 100 mmdishes and grown until they reached ˜70% confluence. Cells were rinsedwith PBS and covered with 4 ml DMEM with 10% fetal calf serum (FCS) andadenoviruses at a MOI of 500. This MOI allow infection of only ˜10% ofthe cells. Cells were incubated at 37° C. with constant but gentleagitation for 6 hours. 6 ml of DMEM with 10% FCS was added and cellswere incubated overnight at 37° C. Cells were then incubated either innormoxia (5% CO2 in normal air at 37° C.) or in hypoxia (5% CO2, 2% O2at 37° C.) for 24 hours.

[0212] RNAses Protection Assay (RPA)

[0213] Total RNA was isolated from transduced cells by Guanidinethiocyanate-phenol extractions as described (Staffa, A., et al., J. BiolChem. (1997) 272: 33394-401) (FIG. 3A or 3C) or with Rneasy Mini Kit™(Qiagen) as described by the manufacturer. 15 μg total RNA per samplewere analyzed by RPA using RPA III kit (Ambion). Probe were preparedfrom hAngio1 template set (Pharmingens) for FIG. 3A or from home madeVEGF and actin template for the others. VEGF template was prepared byamplification on the phVEGF165.SR (from J. F. Isner) (Tsurumi, Y., etal., Circulation. (1996) 94: 3281-3290) with the oligos CCG GM TTC TCTACC TCC ACC ATG CC (SEQ ID NO: 12) and CCG GM TTC CTC AGT GGG CAC ACACTC C (SEQ ID NO: 13), digestion of the amplification product with EcoRI(in both oligos) and insertion in pBluescript cut with EcoRI. Sequenceintegrity was confirmed by sequencing. The resulting plasmid wasdigested with HindIII and transcribed with T7 RNA polymerase to generatean antisense RNA probe. Actin probe template was obtained from W. E.Bradley (Houle, B., et al., Proc. Natl. Aca. Sci. USA (1993) 90:985-989).

[0214] Quantification

[0215] Autoradiograms were scanned on an Alpha Imager 2000™ (AlphaInnotech Corporation). Intensity of each band was measured and relativeexpression was calculated as VEGF intensity/Flt4 intensity for FIG. 3Aor as VEGF intensity/actin intensity for the others. Relative VEGFvalues were then normalized so that a value of 100 was attributed to thecontrol samples in normoxia. Protein VEGF produced were proportional tothe amount of mRNA quantified. Results are expressed as mean±SED.

[0216] Gene Chip Hybridization

[0217] Total RNA was isolated from human SkMC (Clonetics) infected witheither Ad.Null™ (Q-Biogene) or Ad.HIF-2α as described (Staffa, et al.,J. Biol. Chem. (1997) 272: 33394-401). Probes were prepared andhybridized to Atlas Human 1.2 Array™ (Clontech) with ExpressHyb™solution (Clontech) according to the instructions from the manufacturer.The arrays were exposed to phosphorimager screen and analyzed with theAtlas 2.01™ software (Clontech).

[0218] Angiogenesis in Vivo

[0219] Early passage human SkMC (uninfected or infected with theindicated adenovirus vectors) were incorporated into matrigel (50000cells/0.5 ml) and injected subcutaneously into skid mice (10 pergroups). Matrigel alone or bFGF (400 ng/ml) were used as controls. 7days post-implantation, matrigel plugs were excised and processed forhistological evaluation. Each measurement corresponds to the degree ofcellular response in a microscopic field expressed as Integrated Density(sum of the gray values in the selection, with the backgroundsubtracted, 6 measurements on each plug).

[0220] Metabolic activity in infarcted rats created by a permanent leftanterior descending coronary artery ligation (Myoinfarct™ rats, CharlesRiver Laboratories) was measured 5 days post ligature by injection of¹⁸FDG (˜750 μCi/100 g) and dynamic acquisition using a small animalPET-Scan™ (Sherbrooke University). Ten days after the ligature, amini-thoracotomy was performed to inject a solution of Ad.HIF2α/VP16 orsaline (N=2) in the infarcted area of the left ventricle. Another ¹⁸FDGPET-Scan™ was performed 14, 28, 42 and 56 days after the injection.Metabolic activity was calculated as the amount of ¹⁸FDG found in anarea of the lateral wall of the left ventricle, where the infarct islocalized, normalized with the amount of FGD found in an unaffected areain the septal wall that is irrigated by another coronary artery. Afterthe last PET-scan™, animals were sacrificed and blood vessels werequantified in the entire infarcted area or in the fields of the marginsbetween healthy and infarcted tissues.

Results

[0221] In Vitro VEGF Induction by HIF-1α, HIF-2α and HIF-3α

[0222]FIGS. 2A, 2B, 3A, 3B and 3C illustrate the expression ofangiogenesis related genes in human cells transfected with HIFconstructs.

[0223] The natural (wild type) form of HIF factors or activated versionwere introduced in pcDNA3 (Invitrogen Corp.), an expression vector.Activated constructs consist in the deletion of hypoxia regulatedinstability domains located in the N′ part of the proteins and theintroduction of a strong activator of transcription, the VP16 domainfrom Herpes virus (Vincent et al., Circulation (2000) 102: 2255-2261).The resulting hybrids are no longer regulated by hypoxia and alwaysdisplay a transactivation activity.

[0224] These plasmids were transiently transfected in human embryonickidney 293 cells (HEK293) or in human liver cells Hep3B. Cells wereincubated 24 hours either in normoxia (normal conditions) or in hypoxia(2% oxygen) and total RNA was isolated. Similarly, human skeletal musclecells (hSkMC) were transduced with adenovirus vectors expressing some ofthe HIF constructs. Endogenous VEGF gene activation following HIFconstructs transfection was analyzed by RNAse Protection Assay (RPA,Ambion).

[0225] No VEGF induction was produced by HIF-1α transfection in normoxia(similar to control) (FIGS. 2A, 2B, 3A and 3B). This result was expectedsince HIF-1α is quickly degraded in normoxia. The HIF-1α/VP16 hybridstimulated VEGF both in normoxia and hypoxia, since the protein is nolonger regulated by oxygen tension. Interestingly, HIF-3α was veryefficient to stimulate VEGF in HEK293 and Hep3B cells (FIG. 2A and 2B).The levels were superior to those obtained with HIF-1α both in normoxiaand in hypoxia, although HIF-1α/VP16 had an even higher transactivationactivity. The activation of HIF-3α with the VP16 domain did notsignificantly modify the levels of VEGF induction.

[0226] VEGF stimulation by HIF-2α was also very important. FIGS. 3A, 3Band 3C show that the wild type version of the protein stimulated VEGF inranges comparable to HIF-1α/VP16, both in normoxia and in hypoxia, inthe 3 cells types studied (HEK293 in FIG. 3A, Hep3B in FIG. 3B and hSKMCin FIG. 3C). The VP16 fusion did not provide an important advantage inthe levels of VEGF produced.

[0227] Activation of Angiogenic Genes by HIF-2α in Vitro

[0228] VEGF is an important angiogenic gene. However, angiogenesis is acomplex process involving several steps that are regulated by severalfactors. To evaluate HIF factors potential as an angiogenic modulator,gene expression was compared in human SkMC infected either with Ad.HIF2αor Ad.Null™ using gene chip technology. cDNA probes derived from eithercell population was hybridized on a Atlas Human 1.2 Array™ (Clontech)assessing expression of almost 1200 genes. Of the genes differentiallyregulated, VEGF was the one showing the most significant increase. VEGFstimulates both proliferation, migration and differentiation ofendothelial cells. Interestingly, other angiogenic genes were activated(Table 2). As VEGF, Interleukin-8 (IL-8) and the activation of LeukemiaInhibitory Factor Receptor (LIF-R) are known to stimulate theproliferation of endothelial cells. However, IL-8 also acts byactivating the release of metalloproteinases responsible for the basalmembrane disruption, the first step of angiogenesis (FIG. 4). LIF isalso known to enhance survival of SkMC, which would be useful in celltherapy. The role of Interleukin-6 (IL-6) is more indirect: it has noeffect on endothelial cell proliferation, but stimulates their migrationand differentiation. It is also an enhancer of VEGF production.Placental growth factor (PIGF) was shown to act in cooperation with VEGFto stimulate angiogenesis. Matrix metalloproteinase 7 (MMP7) is a potentproteinase able to initiate the angiogenesis process while placentalplasminogen activator inhibitor 2 (PAI-2) is able to stabilize nascentvessels (FIG. 4). There are many other potential angiogenic factors thatcouldn't be detected in this assay because of limitations of the genechip, but that might be induced by HIF factors. TABLE 2 Genes activatedby HIF-2α in SkMC. Gene Fold induction Category VEGF 9.10 Growth factorIL-8 2.26 Growth factor IL-6 2.21 Growth factor PIGF Up* Growth factorLIF-R Up* Growth factor PAI-2 1.93 Proteinase inhibitor MMP7 Up*Metalloproteinase

[0229] HIF-2α Stimulates Angiogenesis in Vivo

[0230] Angiogenic potential of HIF constructs were evaluated in vivo bysubcutaneous implantation of SkMC transfected with HIF constructs inmice. Seven days following implantation, modified cells pellets showedsignificantly higher blood vessel density than unmodified SkMC (FIG.5B). This result confirms that the VP16 activation is not necessary inthe case of HIF-2α. Modified SkMC differentiated in vivo into myotubessupported by extracellular matrix and surrounded by new blood vessels(FIG. 5A). The angiogenesis was important around the myotubes andappeared well organized.

[0231] HIF-2α was also directly delivered in a model of MI in rats.Metabolic activity was assayed 5 days before direct myocardial injectionof the vector Ad.HIF-2α/VP16 and at several time points in the following2 months. As shown in FIGS. 6A and 6B, an improved metabolic activitywas measured in the infarcted area of treated rats over control ones asquantified by position emission tomography-scan (PET-Scan™). When bloodvessel density was quantified by histology, an increase number of bloodvessels was noted for the adenovirus treated rats in the infarcted area,as well as in the peri-infarcted zone (FIG. 6B).

[0232] 3) Discussion

[0233] In vitro transfection experiments showed that wild type HIF-3αand HIF-2α were superior to HIF-1α to induce VEGF. Used in gene therapy,HIF-3α would thus be very useful to modulate angiogenesis.

[0234] As shown in FIGS. 2A, 2B, 3A, 3B and 3C, transfection of HIF-VP16fusion encoding constructs resulted in a significant stimulation ofVEGF. However, in gene therapy in humans, these constructs pose theproblem of immunogenicity. The presence of the VP16 sequence, of viralorigin, is recognized by the immune system and trigger an immuneresponse against the transferred gene. Construct using wild type humangenes, such as HIF-3α, has the advantage of being less immunogenic, thusproviding a longer period of expression. In comparison, HIF-1α had apoor angiogenic potential in normoxia and requires the VP16 modificationto be fully active (FIGS. 2, 3 and Vincent et al., Circulation (2000)102: 2255-2261). FIGS. 2A, 2B, 3A, 3B and 3C also strongly suggest thatHIF-2α and HIF-3α could be used efficiently in gene transfer protocolsto modify gene transcription of the targeted cells. In particular, VEGFexpression is increased, which result in angiogenesis. It is thusproposed to use HIF-2α or HIF-3α sequence in ischemic diseases toincrease blood vessels and blood perfusion.

[0235] The analysis of genes activated by HIF-2α revealed the inductionof several angiogenic genes (Table I). These genes play a role invarious aspects of angiogenesis (FIG. 4) and the resulting angiogenesisis thus expected to be strong and well organized. This is a majoradvantage compared to the use of solely VEGF to induce angiogenesissince all the key steps of angiogenesis will be stimulated. It isexpected that HIF-3α will also regulate several angiogenesis relatedgenes, and possibly the same ones as HIF-2α.

[0236] In vivo experiments proved that HIF-2α gene transfer resulted ina significant angiogenesis, both in sub-cutaneous implants (FIG. 5B) andin a MI heart model (FIG. 6B). These results confirmed that the VP16fusion was not necessary to result in the formation on blood vessels.This characteristic confers an important advantage to HIF-2α sequence asan angiogenic agent. The use of a native sequence in gene transfertherapy will raise a much lower immunogenic response than a constructwith VP16, of viral origin. Since HIF-3α was also superior to HIF-1α andshowed in vitro induction of VEGF similar to HIF-2α, it is expected thatit will also be efficient in vivo.

[0237] Angiogenesis resulted in an increase in metabolic activity(increased glucose consumption) in the infarcted area as shown in FIG.6A, indicating an improvement in the tissue function. This, higher bloodsupply translated in higher muscle activity which can improve heartpumping function.

[0238] In summary, it is clear that HIF factors delivered eitherdirectly or via an implanted cell, offers a great potential formyocardial regeneration and improvement of cardiac function.

[0239] The results reported herein constitute a proof of principle tothe effect that HIF-2α and HIF-3α can be used in gene therapy.

[0240] While several embodiments of the invention have been described,it will be understood that the present invention is capable of furthermodifications, and this application is intended to cover any variations,uses, or adaptations of the invention, following in general theprinciples of the invention and including such departures from thepresent disclosure as to come within knowledge or customary practice inthe art to which the invention pertains, and as may be applied to theessential features hereinbefore set forth and falling within the scopeof the invention or the limits of the appended claims.

1 13 1 2223 DNA Homo sapiens CDS (1)..(2115) 1 atg gac agg tcg acc acggag ctg cgc aag gaa aag tcc cgg gat gcg 48 Met Asp Arg Ser Thr Thr GluLeu Arg Lys Glu Lys Ser Arg Asp Ala 1 5 10 15 gcc cgc agc cgg cgc agccag gag acc gag gtg ctg tac cag ctg gct 96 Ala Arg Ser Arg Arg Ser GlnGlu Thr Glu Val Leu Tyr Gln Leu Ala 20 25 30 cac acg ctg ccc ttc gcc cgcggc gtc agc gcc cac ctg gac aag gcc 144 His Thr Leu Pro Phe Ala Arg GlyVal Ser Ala His Leu Asp Lys Ala 35 40 45 tct atc atg cgc ctc acc atc agctac ctg cgc atg cac cgc ctc tgc 192 Ser Ile Met Arg Leu Thr Ile Ser TyrLeu Arg Met His Arg Leu Cys 50 55 60 gcc gca ggg gag tgg aac cag gtg ggagca ggg gga gaa cca ctg gat 240 Ala Ala Gly Glu Trp Asn Gln Val Gly AlaGly Gly Glu Pro Leu Asp 65 70 75 80 gcc tgc tac ctg aag gcc ctg gag ggcttc gtc atg gtg ctc acc gcc 288 Ala Cys Tyr Leu Lys Ala Leu Glu Gly PheVal Met Val Leu Thr Ala 85 90 95 gag gga gac atg gct tac ctg tcg gag aatgtc agc aaa cac ctg ggc 336 Glu Gly Asp Met Ala Tyr Leu Ser Glu Asn ValSer Lys His Leu Gly 100 105 110 ctc agt cag ctg gag ctc att gga cac agcatc ttt gat ttc atc cac 384 Leu Ser Gln Leu Glu Leu Ile Gly His Ser IlePhe Asp Phe Ile His 115 120 125 ccc tgt gac caa gag gag ctt cag gac gccctg acc ccc caa cag acc 432 Pro Cys Asp Gln Glu Glu Leu Gln Asp Ala LeuThr Pro Gln Gln Thr 130 135 140 ctg tcc agg agg aag gtg gag gcc ccc acggag cgg tgc ttc tcc ttg 480 Leu Ser Arg Arg Lys Val Glu Ala Pro Thr GluArg Cys Phe Ser Leu 145 150 155 160 cgc atg aag agt acg ctc acc agc cgcggg cgc acc ctc aac ctc aag 528 Arg Met Lys Ser Thr Leu Thr Ser Arg GlyArg Thr Leu Asn Leu Lys 165 170 175 gcg gcc acc tgg aag gtg ctg aac tgctct gga cat atg agg gcc tac 576 Ala Ala Thr Trp Lys Val Leu Asn Cys SerGly His Met Arg Ala Tyr 180 185 190 aag cca cct gcg cag act tct cca gctggg agc cct gac tca gag ccc 624 Lys Pro Pro Ala Gln Thr Ser Pro Ala GlySer Pro Asp Ser Glu Pro 195 200 205 ccg ctg cag tgc ccg gtg ctc atc tgcgaa gcc atc ccc cac cca ggc 672 Pro Leu Gln Cys Pro Val Leu Ile Cys GluAla Ile Pro His Pro Gly 210 215 220 agc ctg gag ccc cca ctg ggc cga ggggcc ttc ctc agc cgc cac agc 720 Ser Leu Glu Pro Pro Leu Gly Arg Gly AlaPhe Leu Ser Arg His Ser 225 230 235 240 ctg gac atg aag ttc acc tac tgtgac gac agg att gca gaa gtg gct 768 Leu Asp Met Lys Phe Thr Tyr Cys AspAsp Arg Ile Ala Glu Val Ala 245 250 255 ggc tat agt ccc gat gac ctg atcggc tgt tcc gcc tac gag tac atc 816 Gly Tyr Ser Pro Asp Asp Leu Ile GlyCys Ser Ala Tyr Glu Tyr Ile 260 265 270 cac gcg ctg gac tcc gac gcg gtcagc aag agc atc cac acc ttg ctg 864 His Ala Leu Asp Ser Asp Ala Val SerLys Ser Ile His Thr Leu Leu 275 280 285 agc aag ggc cag gca gta aca gggcag tat cgc ttc ctg gcc cgg agt 912 Ser Lys Gly Gln Ala Val Thr Gly GlnTyr Arg Phe Leu Ala Arg Ser 290 295 300 ggt ggc tac ctg tgg acc cag acccag gcc aca gtg gtg tca ggg gga 960 Gly Gly Tyr Leu Trp Thr Gln Thr GlnAla Thr Val Val Ser Gly Gly 305 310 315 320 cgg ggc ccc cag tcg gag agtatc gtc tgt gtc cat ttt tta atc agc 1008 Arg Gly Pro Gln Ser Glu Ser IleVal Cys Val His Phe Leu Ile Ser 325 330 335 cag gtg gaa gag acc gga gtggtg ctg tcc ctg gag caa acg gag caa 1056 Gln Val Glu Glu Thr Gly Val ValLeu Ser Leu Glu Gln Thr Glu Gln 340 345 350 cac tct cgc aga ccc att cagcgg ggc gcc ccc tct cag aag ggc acc 1104 His Ser Arg Arg Pro Ile Gln ArgGly Ala Pro Ser Gln Lys Gly Thr 355 360 365 cct aac cct ggg gac agc cttgac acc cct ggc ccc cgg atc ctt gcc 1152 Pro Asn Pro Gly Asp Ser Leu AspThr Pro Gly Pro Arg Ile Leu Ala 370 375 380 ttc ctg cac ccg cct tcc ctgagc gag gct gcc ctg gcc gct gac ccc 1200 Phe Leu His Pro Pro Ser Leu SerGlu Ala Ala Leu Ala Ala Asp Pro 385 390 395 400 cgc cgt ttc tgc agc cctgac ctc cgt cgc ctc ctg gga ccc atc ctg 1248 Arg Arg Phe Cys Ser Pro AspLeu Arg Arg Leu Leu Gly Pro Ile Leu 405 410 415 gat ggg gct tca gta gcagcc act ccc agc acc ccg ctg gcc aca cgg 1296 Asp Gly Ala Ser Val Ala AlaThr Pro Ser Thr Pro Leu Ala Thr Arg 420 425 430 cac ccc caa agt cct ctttcg gct gat ctc cca gat gaa cta cct gtg 1344 His Pro Gln Ser Pro Leu SerAla Asp Leu Pro Asp Glu Leu Pro Val 435 440 445 ggc acc gag aat gtg cacaga ctc ttc acc tcc ggg aaa gac act gag 1392 Gly Thr Glu Asn Val His ArgLeu Phe Thr Ser Gly Lys Asp Thr Glu 450 455 460 gca gtg gag aca gat ttagat ata gct cag atg agg aaa ctg aag ctc 1440 Ala Val Glu Thr Asp Leu AspIle Ala Gln Met Arg Lys Leu Lys Leu 465 470 475 480 aga ctg ttg acc acaggc aca gaa ctc aga agt gat ggt gct ggg act 1488 Arg Leu Leu Thr Thr GlyThr Glu Leu Arg Ser Asp Gly Ala Gly Thr 485 490 495 tca gcc aag gtc caccca agt cca agg ctc atc ctc tta cct ccc tcc 1536 Ser Ala Lys Val His ProSer Pro Arg Leu Ile Leu Leu Pro Pro Ser 500 505 510 tgc cct ccg cag gatgct gat gct ctg gat ttg gag atg ctg gcc ccc 1584 Cys Pro Pro Gln Asp AlaAsp Ala Leu Asp Leu Glu Met Leu Ala Pro 515 520 525 tac atc tcc atg gatgat gac ttc cag ctc aac gcc agc gag cag cta 1632 Tyr Ile Ser Met Asp AspAsp Phe Gln Leu Asn Ala Ser Glu Gln Leu 530 535 540 ccc agg gcc tac cacaga cct ctg ggg gct gtc ccc cgg ccc cgt gct 1680 Pro Arg Ala Tyr His ArgPro Leu Gly Ala Val Pro Arg Pro Arg Ala 545 550 555 560 cgg agc ttc catggc ctg tca cct cca gcc ctt gag ccc tcc ctg cta 1728 Arg Ser Phe His GlyLeu Ser Pro Pro Ala Leu Glu Pro Ser Leu Leu 565 570 575 ccc cgc tgg gggagt gac ccc cgg ctg agc tgc tcc agc cct tcc aga 1776 Pro Arg Trp Gly SerAsp Pro Arg Leu Ser Cys Ser Ser Pro Ser Arg 580 585 590 ggg gac ccc tcagca tcc tct ccc atg gct ggg gct cgg aag agg acc 1824 Gly Asp Pro Ser AlaSer Ser Pro Met Ala Gly Ala Arg Lys Arg Thr 595 600 605 ctg gcc cag agctca gag gac gag gac gag gga gtg gag ctg ctg gga 1872 Leu Ala Gln Ser SerGlu Asp Glu Asp Glu Gly Val Glu Leu Leu Gly 610 615 620 gtg aga cct cccaaa agg tcc ccc agc cca gaa cac gaa aac ttt ctg 1920 Val Arg Pro Pro LysArg Ser Pro Ser Pro Glu His Glu Asn Phe Leu 625 630 635 640 ctc ttt cctctc agc ctg agt ttc ctt ctg aca gga gga cca gcc cca 1968 Leu Phe Pro LeuSer Leu Ser Phe Leu Leu Thr Gly Gly Pro Ala Pro 645 650 655 ggg agc ctgcag gac ccc agc acc cca ctc ctg aac ctg aat gag ccc 2016 Gly Ser Leu GlnAsp Pro Ser Thr Pro Leu Leu Asn Leu Asn Glu Pro 660 665 670 ctg ggc ctgggc ccc tca ctg ctc tct ccg tac tca gac gag gac act 2064 Leu Gly Leu GlyPro Ser Leu Leu Ser Pro Tyr Ser Asp Glu Asp Thr 675 680 685 acc cag cccggg ggc ccc ttc cag cca agg gca ggc tca gcc cag gct 2112 Thr Gln Pro GlyGly Pro Phe Gln Pro Arg Ala Gly Ser Ala Gln Ala 690 695 700 gactgagccggct cctctcccca tctgccttct cctcccccag aaaggacctc 2165 Asp 705aaccacactc cacgccggca gccaacgcac aggaggtcct tgccttccgg caccaacg 2223 2705 PRT Homo sapiens 2 Met Asp Arg Ser Thr Thr Glu Leu Arg Lys Glu LysSer Arg Asp Ala 1 5 10 15 Ala Arg Ser Arg Arg Ser Gln Glu Thr Glu ValLeu Tyr Gln Leu Ala 20 25 30 His Thr Leu Pro Phe Ala Arg Gly Val Ser AlaHis Leu Asp Lys Ala 35 40 45 Ser Ile Met Arg Leu Thr Ile Ser Tyr Leu ArgMet His Arg Leu Cys 50 55 60 Ala Ala Gly Glu Trp Asn Gln Val Gly Ala GlyGly Glu Pro Leu Asp 65 70 75 80 Ala Cys Tyr Leu Lys Ala Leu Glu Gly PheVal Met Val Leu Thr Ala 85 90 95 Glu Gly Asp Met Ala Tyr Leu Ser Glu AsnVal Ser Lys His Leu Gly 100 105 110 Leu Ser Gln Leu Glu Leu Ile Gly HisSer Ile Phe Asp Phe Ile His 115 120 125 Pro Cys Asp Gln Glu Glu Leu GlnAsp Ala Leu Thr Pro Gln Gln Thr 130 135 140 Leu Ser Arg Arg Lys Val GluAla Pro Thr Glu Arg Cys Phe Ser Leu 145 150 155 160 Arg Met Lys Ser ThrLeu Thr Ser Arg Gly Arg Thr Leu Asn Leu Lys 165 170 175 Ala Ala Thr TrpLys Val Leu Asn Cys Ser Gly His Met Arg Ala Tyr 180 185 190 Lys Pro ProAla Gln Thr Ser Pro Ala Gly Ser Pro Asp Ser Glu Pro 195 200 205 Pro LeuGln Cys Pro Val Leu Ile Cys Glu Ala Ile Pro His Pro Gly 210 215 220 SerLeu Glu Pro Pro Leu Gly Arg Gly Ala Phe Leu Ser Arg His Ser 225 230 235240 Leu Asp Met Lys Phe Thr Tyr Cys Asp Asp Arg Ile Ala Glu Val Ala 245250 255 Gly Tyr Ser Pro Asp Asp Leu Ile Gly Cys Ser Ala Tyr Glu Tyr Ile260 265 270 His Ala Leu Asp Ser Asp Ala Val Ser Lys Ser Ile His Thr LeuLeu 275 280 285 Ser Lys Gly Gln Ala Val Thr Gly Gln Tyr Arg Phe Leu AlaArg Ser 290 295 300 Gly Gly Tyr Leu Trp Thr Gln Thr Gln Ala Thr Val ValSer Gly Gly 305 310 315 320 Arg Gly Pro Gln Ser Glu Ser Ile Val Cys ValHis Phe Leu Ile Ser 325 330 335 Gln Val Glu Glu Thr Gly Val Val Leu SerLeu Glu Gln Thr Glu Gln 340 345 350 His Ser Arg Arg Pro Ile Gln Arg GlyAla Pro Ser Gln Lys Gly Thr 355 360 365 Pro Asn Pro Gly Asp Ser Leu AspThr Pro Gly Pro Arg Ile Leu Ala 370 375 380 Phe Leu His Pro Pro Ser LeuSer Glu Ala Ala Leu Ala Ala Asp Pro 385 390 395 400 Arg Arg Phe Cys SerPro Asp Leu Arg Arg Leu Leu Gly Pro Ile Leu 405 410 415 Asp Gly Ala SerVal Ala Ala Thr Pro Ser Thr Pro Leu Ala Thr Arg 420 425 430 His Pro GlnSer Pro Leu Ser Ala Asp Leu Pro Asp Glu Leu Pro Val 435 440 445 Gly ThrGlu Asn Val His Arg Leu Phe Thr Ser Gly Lys Asp Thr Glu 450 455 460 AlaVal Glu Thr Asp Leu Asp Ile Ala Gln Met Arg Lys Leu Lys Leu 465 470 475480 Arg Leu Leu Thr Thr Gly Thr Glu Leu Arg Ser Asp Gly Ala Gly Thr 485490 495 Ser Ala Lys Val His Pro Ser Pro Arg Leu Ile Leu Leu Pro Pro Ser500 505 510 Cys Pro Pro Gln Asp Ala Asp Ala Leu Asp Leu Glu Met Leu AlaPro 515 520 525 Tyr Ile Ser Met Asp Asp Asp Phe Gln Leu Asn Ala Ser GluGln Leu 530 535 540 Pro Arg Ala Tyr His Arg Pro Leu Gly Ala Val Pro ArgPro Arg Ala 545 550 555 560 Arg Ser Phe His Gly Leu Ser Pro Pro Ala LeuGlu Pro Ser Leu Leu 565 570 575 Pro Arg Trp Gly Ser Asp Pro Arg Leu SerCys Ser Ser Pro Ser Arg 580 585 590 Gly Asp Pro Ser Ala Ser Ser Pro MetAla Gly Ala Arg Lys Arg Thr 595 600 605 Leu Ala Gln Ser Ser Glu Asp GluAsp Glu Gly Val Glu Leu Leu Gly 610 615 620 Val Arg Pro Pro Lys Arg SerPro Ser Pro Glu His Glu Asn Phe Leu 625 630 635 640 Leu Phe Pro Leu SerLeu Ser Phe Leu Leu Thr Gly Gly Pro Ala Pro 645 650 655 Gly Ser Leu GlnAsp Pro Ser Thr Pro Leu Leu Asn Leu Asn Glu Pro 660 665 670 Leu Gly LeuGly Pro Ser Leu Leu Ser Pro Tyr Ser Asp Glu Asp Thr 675 680 685 Thr GlnPro Gly Gly Pro Phe Gln Pro Arg Ala Gly Ser Ala Gln Ala 690 695 700 Asp705 3 2115 DNA Homo sapiens 3 atggacaggt cgaccacgga gctgcgcaaggaaaagtccc gggatgcggc ccgcagccgg 60 cgcagccagg agaccgaggt gctgtaccagctggctcaca cgctgccctt cgcccgcggc 120 gtcagcgccc acctggacaa ggcctctatcatgcgcctca ccatcagcta cctgcgcatg 180 caccgcctct gcgccgcagg ggagtggaaccaggtgggag cagggggaga accactggat 240 gcctgctacc tgaaggccct ggagggcttcgtcatggtgc tcaccgccga gggagacatg 300 gcttacctgt cggagaatgt cagcaaacacctgggcctca gtcagctgga gctcattgga 360 cacagcatct ttgatttcat ccacccctgtgaccaagagg agcttcagga cgccctgacc 420 ccccaacaga ccctgtccag gaggaaggtggaggccccca cggagcggtg cttctccttg 480 cgcatgaaga gtacgctcac cagccgcgggcgcaccctca acctcaaggc ggccacctgg 540 aaggtgctga actgctctgg acatatgagggcctacaagc cacctgcgca gacttctcca 600 gctgggagcc ctgactcaga gcccccgctgcagtgcccgg tgctcatctg cgaagccatc 660 ccccacccag gcagcctgga gcccccactgggccgagggg ccttcctcag ccgccacagc 720 ctggacatga agttcaccta ctgtgacgacaggattgcag aagtggctgg ctatagtccc 780 gatgacctga tcggctgttc cgcctacgagtacatccacg cgctggactc cgacgcggtc 840 agcaagagca tccacacctt gctgagcaagggccaggcag taacagggca gtatcgcttc 900 ctggcccgga gtggtggcta cctgtggacccagacccagg ccacagtggt gtcaggggga 960 cggggccccc agtcggagag tatcgtctgtgtccattttt taatcagcca ggtggaagag 1020 accggagtgg tgctgtccct ggagcaaacggagcaacact ctcgcagacc cattcagcgg 1080 ggcgccccct ctcagaaggg cacccctaaccctggggaca gccttgacac ccctggcccc 1140 cggatccttg ccttcctgca cccgccttccctgagcgagg ctgccctggc cgctgacccc 1200 cgccgtttct gcagccctga cctccgtcgcctcctgggac ccatcctgga tggggcttca 1260 gtagcagcca ctcccagcac cccgctggccacacggcacc cccaaagtcc tctttcggct 1320 gatctcccag atgaactacc tgtgggcaccgagaatgtgc acagactctt cacctccggg 1380 aaagacactg aggcagtgga gacagatttagatatagctc agatgaggaa actgaagctc 1440 agactgttga ccacaggcac agaactcagaagtgatggtg ctgggacttc agccaaggtc 1500 cacccaagtc caaggctcat cctcttacctccctcctgcc ctccgcagga tgctgatgct 1560 ctggatttgg agatgctggc cccctacatctccatggatg atgacttcca gctcaacgcc 1620 agcgagcagc tacccagggc ctaccacagacctctggggg ctgtcccccg gccccgtgct 1680 cggagcttcc atggcctgtc acctccagcccttgagccct ccctgctacc ccgctggggg 1740 agtgaccccc ggctgagctg ctccagcccttccagagggg acccctcagc atcctctccc 1800 atggctgggg ctcggaagag gaccctggcccagagctcag aggacgagga cgagggagtg 1860 gagctgctgg gagtgagacc tcccaaaaggtcccccagcc cagaacacga aaactttctg 1920 ctctttcctc tcagcctgag tttccttctgacaggaggac cagccccagg gagcctgcag 1980 gaccccagca ccccactcct gaacctgaatgagcccctgg gcctgggccc ctcactgctc 2040 tctccgtact cagacgagga cactacccagcccgggggcc ccttccagcc aagggcaggc 2100 tcagcccagg ctgac 2115 4 21 DNAArtificial sequence Synthetic oligonucleotide 4 ttaagatatc gatgacacgt g21 5 21 DNA Artificial sequence Synthetic oligonucleotide 5 tcagcacgtgtcatcgatat c 21 6 24 DNA Artificial sequence Synthetic oligonucleotide 6gctagctagg aagttactcc tctc 24 7 33 DNA Artificial sequence Syntheticoligonucleotide 7 ccatggacag gtcgaccacg gagctgcgca agg 33 8 25 DNAArtificial sequence Synthetic oligonucleotide 8 cgcaggcagg tggcttgtaggccct 25 9 27 DNA Artificial sequence Synthetic oligonucleotide 9cagctggagc tcattggaca cagcatc 27 10 24 DNA Artificial sequence Syntheticoligonucleotide 10 ccccatcctg tgcgttggct gccg 24 11 28 DNA Artificialsequence Synthetic oligonucleotide 11 ggagtcagct taagctgaat gggtctgc 2812 26 DNA Artificial sequence Synthetic oligonucleotide 12 ccggaattctctacctccac catgcc 26 13 28 DNA Artificial sequence Syntheticoligonucleotide 13 ccggaattcc tcagtgggca cacactcc 28

What is claimed is:
 1. An isolated or purified nucleic acid molecule comprising a sequence selected from the group consisting of: a) sequences provided in SEQ ID NO: 1 or 3; b) complements of the sequences provided in SEQ ID NO: 1 or 3; c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NO: 1 or 3; d) sequences that hybridize to a sequence provided in SEQ ID NO: 1 or 3, under moderately stringent conditions; e) sequences having at least 75% identity to a sequence of SEQ ID NO: 1 or 3; and f) degenerate variants of a sequence provided in SEQ ID NO: 1 or
 3. 2. The nucleic acid of claim 1, wherein it comprises a sequence selected from the group consisting of: a) a nucleotide sequence having at least 75% nucleotide sequence identity with SEQ ID NO: 1; and b) a nucleotide sequence having at least 75% nucleotide sequence identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO:
 2. 3. The nucleic acid of claim 2, wherein it comprises a sequence selected from the group consisting of. a) a sequence substantially the same as SEQ ID NO: 1; and b) a sequence substantially the same as a nucleic acid encoding an amino acid sequence of SEQ ID NO:
 2. 4. The nucleic acid of claim 3, wherein it comprises a sequence selected from the group consisting of: a) a sequence having 100% identity with SEQ ID NO: 1; b) a sequence having 100% identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO:
 2. 5. The nucleic acid molecule of claim 1, wherein it comprises: a) part or all of nucleic acids 1423 to 1545 of SEQ ID NO: 1; b) part or all of nucleic acids encoding a polypeptide with amino acids 475 to 515 of SEQ ID NO:
 2. 6. The nucleic acid molecule of claim 1, wherein it encodes a polypeptide having the biological activity of a human HIF-3α polypeptide.
 7. An isolated or purified nucleic acid molecule comprising a sequence encoding a human HIF-3α polypeptide or degenerate variants thereof, wherein said human HIF-3α polypeptide or degenerate variant comprises part or all of amino acids 475 to 515 of SEQ ID NO:
 2. 8. The nucleic acid molecule of claim 7, wherein it comprises part or all of nucleic acids 1423 to 1545 of SEQ ID NO:
 1. 9. The nucleic acid molecule of claim 7, wherein it comprises SEQ ID NO:
 1. 10. The nucleic acid of claim 7, wherein said nucleic acid molecule consists of a cDNA.
 11. An isolated or purified nucleic acid molecule which hybridizes under high stringency conditions with part or all of any of the nucleic acid molecules of claim 7 or with part or all of a complementary sequence thereof.
 12. The nucleic acid molecule of claim 11, wherein it hybridizes under moderate or high stringency conditions with part or all of nucleic acids 1423 to 1545 of SEQ ID NO: 1, or with part or all of a complementary sequence thereof.
 13. An isolated or purified protein comprising an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid of claim 1; b) sequences having at least 80% identity to SEQ ID NO: 2; c) sequences having at least 85% homology to SEQ ID NO: 2; d) sequence provided in SEQ ID NO: 2; e) sequences having at least 80% identity to amino acid sequences encoded by an open reading frame having SEQ ID NO: 3; and f) sequences having at least 85% sequence homology to amino acid sequences encoded by an open reading frame having SEQ ID NO:
 3. 14. The protein of claim 13, wherein it comprises an amino acid sequence selected from the group consisting of: a) sequences substantially the same as SEQ ID NO: 2; and b) sequences substantially the same as amino acid sequences encoded by an open reading frame having SEQ ID NO:
 3. 15. The protein of claim 14, wherein it comprises an amino acid sequence 100% identical to SEQ ID NO:
 2. 16. The protein of claim 13, wherein said protein has the biological activity of a human HIF-3α polypeptide.
 17. The protein of claim 16, wherein said biological activity comprises inducing VEGF expression.
 18. The protein of claim 17, wherein inducement of VEGF expression promotes angiogenesis.
 19. An isolated or purified human protein comprising part or all of amino acids 475 to 515 of SEQ ID NO: 2, or degenerate variants thereof.
 20. The human protein of claim 19, wherein said protein has the biological activity of a human HIF-3α polypeptide.
 21. The protein of claim 20, wherein said biological activity comprises inducing VEGF expression.
 22. An antisense nucleic acid that reduces a human HIF-3α polypeptide levels of expression.
 23. The antisense of claim 22, wherein said human HIF-3α polypeptide comprise amino acids 475 to 515 of SEQ ID NO:
 2. 24. The antisense of claim 23, wherein said human HIF-3α polypeptide is encoded by an open reading frame having SEQ ID NO:
 3. 25. The antisense of claim 22, wherein said antisense hybridizes under high stringency condition with part or all of SEQ ID NO: 1, or with part or all of a complementary sequence thereof.
 26. The antisense nucleic acid of claim 25, wherein said antisense hybridizes under high stringency conditions with part or all of nucleic acids 1423 to 1545 of SEQ ID NO: 1, or with part or all of a complementary sequence thereof.
 27. The antisense nucleic acid of claim 26, wherein said antisense hybridizes under high stringency conditions with part or all of nucleic acids 2116 to 2223 of SEQ ID NO: 1, or with part or all of a complementary sequence thereof.
 28. The antisense of claim 22, wherein said antisense hybridizes under high stringency conditions to a genomic sequence or to a mRNA.
 29. An isolated or purified antibody that specifically binds to a HIF-3α polypeptide substantially the same as SEQ ID NO:
 2. 30. The antibody of claim 29, wherein said antibody specifically binds to a HIF-3α polypeptide comprising part or all of amino acids 475 to 515 of SEQ ID NO:
 2. 31. The antibody of claim 29, wherein said antibody specifically binds to part or all of amino acids 475 to 515 of SEQ ID NO:
 2. 32. The antibody of claim 29, wherein said antibody consists of a monoclonal or of a polyclonal antibody.
 33. A pharmaceutical composition comprising: 1) at least one element selected from the group consisting of: i) a nucleic acid molecule encoding a human HIF-3α polypeptide; ii) a human HIF-3α polypeptide; iii) an antisense nucleic acid that reduces a human HIF-3α polypeptide levels of expression; and iv) an isolated or purified antibody that specifically binds to a HIF-3α polypeptide; and 2) a pharmaceutically acceptable carrier.
 34. The composition of claim 33, wherein said human HIF-3α polypeptide has an amino acid sequence substantially the same as SEQ ID NO:
 2. 35. The composition of claim 33, wherein said human HIF-3α polypeptide comprises amino acids 475 to 515 of SEQ ID NO:−2.
 36. A cloning or expression vector comprising the nucleic acid of claim
 1. 37. The vector of claim 36, wherein said vector is capable of directing expression of the peptide encoded by said nucleic acid in a vector-containing cell.
 38. The vector of claim 37, wherein said vector is selected from the group consisting of plasmids, adenoviruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses.
 39. A transformed or transfected cell that contains the nucleic acid of claim
 1. 40. The cell of claim 39, wherein said cell consists of a cell selected from the group consisting of HEK293 cells, Hep3B cells, mammalian skeletal muscular cells, cardiac cells, bone marrow cells, fibroblasts, smooth muscle cells, endothelial cells, endothelial progenitor cells and embryonic stem cells.
 41. A transgenic animal generated from the cell of claim 39, wherein said nucleic acid is expressed in said transgenic animal.
 42. A nucleotide probe comprising a sequence of at least 15 sequential nucleotides of SEQ ID NO: 1 or of a sequence complementary to SEQ ID NO:
 1. 43. The nucleotide probe of claim 42, comprising part or all of nucleic acids 1423 to 1545 of SEQ ID NO: 1, part or all of nucleic acids 2116 to 2223 of SEQ ID NO: 1, part or all of nucleic acids encoding amino acids 475 to 515 of SEQ ID NO: 2, or part or all of a complementary sequence thereof.
 44. A method for inducing VEGF expression in a mammalian cell, the method comprising step of introducing and expressing in said cell a nucleic acid sequence encoding polypeptide having the biological activity of a human HIF-3α polypeptide.
 45. The method of claim 44, wherein said human HIF-3α polypeptide comprises an amino acid sequence substantially the same as SEQ ID NO:
 2. 46. The method of claim 45, wherein said human HIF-3α polypeptide comprises part or all of amino acids 475 to 515 of SEQ ID NO:
 2. 47. The method of claim 45, wherein a nucleic acid sequence comprises part or all of nucleic acids 1423 to 1545 of SEQ ID NO:
 1. 48. The method of claim 44, wherein said cell consists of a cardiac cell located in the heart of a living mammal, and wherein expression of said polypeptide induce angiogenesis in cardiac tissue of said mammal.
 49. The method of claim 44, wherein said cell consists of a muscular cell located in muscular tissue of a living mammal, and wherein expression of said polypeptide induce angiogenesis in the muscular tissue of said mammal.
 50. The method of claim 44, further comprising the step of transplanting said cell in tissue of a compatible mammalian recipient.
 51. The method of claim 50, wherein said cells are transplanted in an amount that is sufficient to induce angiogenesis locally or in surrounding transplanted tissue.
 52. The method of claim 51, wherein said transplanted tissue consists of an ischemic or a non-ischemic tissue.
 53. The method of claim 44, wherein said mammalian cell is a skeletal muscular cell thereby providing a HIF-3α expressing-skeletal muscular cell, and wherein said method further comprises the step of transplanting a plurality of said HIF-3α expressing-skeletal muscular cells in a cardiac tissue of a compatible mammalian recipient.
 54. The method of claim 53, wherein the transplantation step consists of an autologous transplantation and wherein said mammalian recipient is a human.
 55. A method for inducing angiogenesis in a mammalian tissue having a plurality of cells, the method comprising the step of introducing and expressing in at least some of said cells a nucleic acid sequence encoding a polypeptide having the biological activity of a human HIF-3α polypeptide.
 56. The method of claim 55, wherein said human HIF-3α polypeptide comprises an amino acid sequence substantially the same as SEQ ID NO:
 2. 57. The method of claim 56, wherein said human HIF-3α polypeptide comprises part or all of amino acids 475 to 515 of SEQ ID NO:
 2. 58. The method of claim 56, wherein said nucleic acid sequence comprises part or all of nucleic acids 1423 to 1545 of SEQ ID NO:
 1. 59. A method for modulating tumoral cell survival or for eliminating a tumoral cell in a mammal, comprising the step of reducing cellular expression levels of a HIF-3α polypeptide.
 60. The method of claim 59, wherein said mammal consists of a human, and wherein the method comprises delivering a human HIF-3α antisense into said tumoral cell.
 61. A method for determining the amount of a human HIF-3α polypeptide or a human HIF-3α nucleic acid in a biological sample, comprising the step of contacting said sample with the antibody of claim 29 or with a probe according to claim
 42. 62. A method of evaluating malignancy of a tumor in a human subject, comprising the step of determining the amount of a HIF-3α polypeptide or of a HIF-3α nucleic acid in a tumoral cell from said subject, wherein said amount is indicative of a degree of malignancy for said tumor.
 63. A kit for determining the amount of a human HIF-3α polypeptide or a human HIF-3α nucleic acid in a biological sample, said kit comprising the antibody of claim 29 or the probe according to claim 42, and at least one element selected from the group consisting of instructions for using said kit, reaction buffer(s), and enzyme(s).
 64. A method for producing a human HIF-3α polypeptide comprising: providing a cell transformed with a nucleic acid sequence encoding a human HIF-3α polypeptide positioned for expression in said cell; culturing said transformed cell under conditions suitable for expressing said nucleic acid; and producing said human HIF-3α polypeptide. 